CA2271132C - Biocompatible compositions - Google Patents
Biocompatible compositions Download PDFInfo
- Publication number
- CA2271132C CA2271132C CA002271132A CA2271132A CA2271132C CA 2271132 C CA2271132 C CA 2271132C CA 002271132 A CA002271132 A CA 002271132A CA 2271132 A CA2271132 A CA 2271132A CA 2271132 C CA2271132 C CA 2271132C
- Authority
- CA
- Canada
- Prior art keywords
- alkylene
- group
- heparin
- polymer
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000000203 mixture Substances 0.000 title claims description 26
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 106
- 229920000669 heparin Polymers 0.000 claims abstract description 105
- 229960002897 heparin Drugs 0.000 claims abstract description 105
- 239000000178 monomer Substances 0.000 claims abstract description 81
- 229920000642 polymer Polymers 0.000 claims abstract description 73
- 239000000758 substrate Substances 0.000 claims abstract description 30
- 229920001897 terpolymer Polymers 0.000 claims abstract description 29
- 229920002683 Glycosaminoglycan Polymers 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 239000008199 coating composition Substances 0.000 claims abstract description 3
- 125000002091 cationic group Chemical group 0.000 claims description 48
- 238000000576 coating method Methods 0.000 claims description 21
- 229910052739 hydrogen Inorganic materials 0.000 claims description 21
- 125000002947 alkylene group Chemical group 0.000 claims description 19
- 239000002904 solvent Substances 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- 125000001153 fluoro group Chemical group F* 0.000 claims description 15
- 239000001257 hydrogen Substances 0.000 claims description 13
- 229920006296 quaterpolymer Polymers 0.000 claims description 13
- -1 siloxane substituent Chemical group 0.000 claims description 13
- 125000000217 alkyl group Chemical group 0.000 claims description 12
- 125000000129 anionic group Chemical group 0.000 claims description 12
- 238000004132 cross linking Methods 0.000 claims description 11
- 125000000962 organic group Chemical group 0.000 claims description 11
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- 125000003118 aryl group Chemical group 0.000 claims description 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 6
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- 125000000623 heterocyclic group Chemical group 0.000 claims description 4
- 125000001165 hydrophobic group Chemical group 0.000 claims description 4
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- 102000007625 Hirudins Human genes 0.000 claims description 2
- 108010007267 Hirudins Proteins 0.000 claims description 2
- 229940072056 alginate Drugs 0.000 claims description 2
- 229920000615 alginic acid Polymers 0.000 claims description 2
- 235000010443 alginic acid Nutrition 0.000 claims description 2
- 125000002877 alkyl aryl group Chemical group 0.000 claims description 2
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 2
- 229910052731 fluorine Inorganic materials 0.000 claims description 2
- 239000011737 fluorine Chemical group 0.000 claims description 2
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 claims description 2
- 229940006607 hirudin Drugs 0.000 claims description 2
- 229920002674 hyaluronan Polymers 0.000 claims description 2
- 229960003160 hyaluronic acid Drugs 0.000 claims description 2
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 claims 4
- 102100022631 Glutamate receptor ionotropic, NMDA 2C Human genes 0.000 claims 3
- 108091008646 testicular receptors Proteins 0.000 claims 3
- 239000004254 Ammonium phosphate Substances 0.000 claims 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims 1
- 235000019289 ammonium phosphates Nutrition 0.000 claims 1
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 claims 1
- 239000011203 carbon fibre reinforced carbon Substances 0.000 claims 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical group [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims 1
- 125000004185 ester group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 21
- 210000004369 blood Anatomy 0.000 abstract description 19
- 239000008280 blood Substances 0.000 abstract description 19
- GMSCBRSQMRDRCD-UHFFFAOYSA-N dodecyl 2-methylprop-2-enoate Chemical compound CCCCCCCCCCCCOC(=O)C(C)=C GMSCBRSQMRDRCD-UHFFFAOYSA-N 0.000 abstract description 9
- 229910019142 PO4 Inorganic materials 0.000 abstract description 5
- 239000010452 phosphate Substances 0.000 abstract description 5
- 150000003839 salts Chemical class 0.000 abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 4
- GYXOHPLRXDQGBP-UHFFFAOYSA-M 2-hydroxyethyl(trimethyl)azanium;2-methylprop-2-enoate Chemical compound CC(=C)C([O-])=O.C[N+](C)(C)CCO GYXOHPLRXDQGBP-UHFFFAOYSA-M 0.000 abstract description 3
- 230000002209 hydrophobic effect Effects 0.000 abstract description 3
- 150000001768 cations Chemical class 0.000 abstract 1
- 239000002607 heparin antagonist Substances 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 24
- 239000000243 solution Substances 0.000 description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical class OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 23
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 22
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 22
- 239000002953 phosphate buffered saline Substances 0.000 description 22
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 20
- 239000011248 coating agent Substances 0.000 description 18
- 239000011541 reaction mixture Substances 0.000 description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 102000008946 Fibrinogen Human genes 0.000 description 11
- 108010049003 Fibrinogen Proteins 0.000 description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- 229940012952 fibrinogen Drugs 0.000 description 10
- 239000003999 initiator Substances 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 9
- JMMWKPVZQRWMSS-UHFFFAOYSA-N isopropanol acetate Natural products CC(C)OC(C)=O JMMWKPVZQRWMSS-UHFFFAOYSA-N 0.000 description 9
- 229940011051 isopropyl acetate Drugs 0.000 description 9
- GWYFCOCPABKNJV-UHFFFAOYSA-N isovaleric acid Chemical compound CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 239000011521 glass Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 229910052698 phosphorus Inorganic materials 0.000 description 7
- 239000011877 solvent mixture Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 210000001772 blood platelet Anatomy 0.000 description 5
- 229920001577 copolymer Polymers 0.000 description 5
- 229910001873 dinitrogen Inorganic materials 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 230000010412 perfusion Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 5
- 230000002965 anti-thrombogenic effect Effects 0.000 description 4
- 239000003146 anticoagulant agent Substances 0.000 description 4
- 229940127219 anticoagulant drug Drugs 0.000 description 4
- 239000006260 foam Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 229960004592 isopropanol Drugs 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- PZJJKWKADRNWSW-UHFFFAOYSA-N trimethoxysilicon Chemical group CO[Si](OC)OC PZJJKWKADRNWSW-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 101150041968 CDC13 gene Proteins 0.000 description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 3
- 239000004743 Polypropylene Substances 0.000 description 3
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 125000004429 atom Chemical group 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- UXYBXUYUKHUNOM-UHFFFAOYSA-M ethyl(trimethyl)azanium;chloride Chemical compound [Cl-].CC[N+](C)(C)C UXYBXUYUKHUNOM-UHFFFAOYSA-M 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000002480 mineral oil Substances 0.000 description 3
- 235000010446 mineral oil Nutrition 0.000 description 3
- 238000011056 performance test Methods 0.000 description 3
- 230000002572 peristaltic effect Effects 0.000 description 3
- 239000004417 polycarbonate Substances 0.000 description 3
- 229920001155 polypropylene Polymers 0.000 description 3
- 239000012265 solid product Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- GNSFRPWPOGYVLO-UHFFFAOYSA-N 3-hydroxypropyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCCO GNSFRPWPOGYVLO-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 239000004593 Epoxy Chemical group 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 150000003926 acrylamides Chemical class 0.000 description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 2
- 229920006317 cationic polymer Polymers 0.000 description 2
- 239000003093 cationic surfactant Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- ZJXZSIYSNXKHEA-UHFFFAOYSA-N ethyl dihydrogen phosphate Chemical compound CCOP(O)(O)=O ZJXZSIYSNXKHEA-UHFFFAOYSA-N 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 125000005843 halogen group Chemical group 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 125000003010 ionic group Chemical group 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen(.) Chemical compound [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000005580 one pot reaction Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical group [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- QLOKJRIVRGCVIM-UHFFFAOYSA-N 1-[(4-methylsulfanylphenyl)methyl]piperazine Chemical compound C1=CC(SC)=CC=C1CN1CCNCC1 QLOKJRIVRGCVIM-UHFFFAOYSA-N 0.000 description 1
- LUYAMNYBNTVQJG-UHFFFAOYSA-N 1-chloro-2-(2-chloroethylsulfonyl)ethane Chemical compound ClCCS(=O)(=O)CCCl LUYAMNYBNTVQJG-UHFFFAOYSA-N 0.000 description 1
- XFGANBYCJWQYBI-UHFFFAOYSA-N 11-bromoundecan-1-ol Chemical compound OCCCCCCCCCCCBr XFGANBYCJWQYBI-UHFFFAOYSA-N 0.000 description 1
- FIYLNTKHSCVZBK-UHFFFAOYSA-N 2-(2,5-dioxopyrrolidin-1-yl)-4-methylbenzenesulfonic acid Chemical compound CC1=CC(=C(C=C1)S(=O)(=O)O)N2C(=O)CCC2=O FIYLNTKHSCVZBK-UHFFFAOYSA-N 0.000 description 1
- XDLMVUHYZWKMMD-UHFFFAOYSA-N 3-trimethoxysilylpropyl 2-methylprop-2-enoate Chemical compound CO[Si](OC)(OC)CCCOC(=O)C(C)=C XDLMVUHYZWKMMD-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241000406799 Deto Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229910003873 O—P—O Inorganic materials 0.000 description 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical compound OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004820 Pressure-sensitive adhesive Substances 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- KXKVLQRXCPHEJC-UHFFFAOYSA-N acetic acid trimethyl ester Natural products COC(C)=O KXKVLQRXCPHEJC-UHFFFAOYSA-N 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
- 229920006243 acrylic copolymer Polymers 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 125000005250 alkyl acrylate group Chemical group 0.000 description 1
- 125000005275 alkylenearyl group Chemical group 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- SWLVFNYSXGMGBS-UHFFFAOYSA-N ammonium bromide Chemical compound [NH4+].[Br-] SWLVFNYSXGMGBS-UHFFFAOYSA-N 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 229910001423 beryllium ion Inorganic materials 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 125000000490 cinnamyl group Chemical group C(C=CC1=CC=CC=C1)* 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000011243 crosslinked material Substances 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000012812 general test Methods 0.000 description 1
- 125000003055 glycidyl group Chemical group C(C1CO1)* 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 229940106780 human fibrinogen Drugs 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- VHRYZQNGTZXDNX-UHFFFAOYSA-N methacryloyl chloride Chemical compound CC(=C)C(Cl)=O VHRYZQNGTZXDNX-UHFFFAOYSA-N 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- XYFCBTPGUUZFHI-UHFFFAOYSA-O phosphonium Chemical compound [PH4+] XYFCBTPGUUZFHI-UHFFFAOYSA-O 0.000 description 1
- 125000005496 phosphonium group Chemical group 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000139 polyethylene terephthalate Polymers 0.000 description 1
- 239000005020 polyethylene terephthalate Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 125000005373 siloxane group Chemical group [SiH2](O*)* 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 125000003808 silyl group Chemical group [H][Si]([H])([H])[*] 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000008259 solid foam Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000012258 stirred mixture Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical compound OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-O sulfonium Chemical compound [SH3+] RWSOTUBLDIXVET-UHFFFAOYSA-O 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 125000005369 trialkoxysilyl group Chemical group 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- RRHXZLALVWBDKH-UHFFFAOYSA-M trimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]azanium;chloride Chemical compound [Cl-].CC(=C)C(=O)OCC[N+](C)(C)C RRHXZLALVWBDKH-UHFFFAOYSA-M 0.000 description 1
- KRLHYNPADOCLAJ-UHFFFAOYSA-N undecyl 2-methylprop-2-enoate Chemical compound CCCCCCCCCCCOC(=O)C(C)=C KRLHYNPADOCLAJ-UHFFFAOYSA-N 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 125000002348 vinylic group Chemical group 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- PAPBSGBWRJIAAV-UHFFFAOYSA-N ε-Caprolactone Chemical compound O=C1CCCCCO1 PAPBSGBWRJIAAV-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
- A61L33/0029—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate using an intermediate layer of polymer
Landscapes
- Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Materials Engineering (AREA)
- Chemical & Material Sciences (AREA)
- Surgery (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Materials For Medical Uses (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Manufacture Of Macromolecular Shaped Articles (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
A polymer is formed from monomers including a zwitterionic monomer, a cation ic monomer and a hydrophobic termonomer. The polymer may be cross-linkable. The polymer may be coated onto a surface from a liquid coating composition followed by contacting the coated surface with heparin. The double coated substrates have improved heparin activity over extended periods as compared to heparinised surfaces which are commercially available. Other anionically charged mucopolysaccharides may be used. The polymers may also scavenge heparin from treated blood to avoid use of potentially toxic heparin inhibitors. Preferably the terpolymer is formed from 2-methacryloyloxyethyl-2-(trimethylamonium)phosphate inner salt, choline methacrylate and n-dodecyl methacrylate.
Description
BIOCOMPATIBLE COMPOSITIONS
The present invention relates to novel biocompatible polymers, the use of these polymers to improve binding of mucopolysaccharides to substrates to improve their hemocompatibility and compositions containing mucopolysaccharides and the polymer.
In WO-A-93/01221 we describe various polymers and their use to coat surfaces to improve their biocompatibility. The polymers include zwitterionic groups ZO and pendant groups which are capable of providing stable surface binding of the polymer to underlying substrate surfaces. The binding may be by provision of pendant hydrophobic groups which physisorb onto hydrophobic substrates, by counterionic attraction between pendant ionic groups on the polymer and oppositely charged groups at the substrate surface, by providing covalent attachment between coreactive pendant groups on the polymer and groups at the substrate surface or by crosslinking the polymer after coating. Post coating crosslinking may also be used to improve the stability of a polymer which is physisorbed, covalently bonded or counterionically bonded to the surface. The polymers have good hemocompatibility as indicated by the low platelet adhesion values reported in that specification.
It has also been shown that zwitterionic groups at substrate surfaces, for instance of contact lenses, show lower rates of deposition of proteins and lipids from biological liquids such as tear film. In WO-A-92/07885, reduced levels of protein deposition are described for contact lenses formed from a hydrogel of a crosslinked copolymer of copolymerisable zwitterionic monomer and non ionic comonomer.
In WO-A-93/21970 it is disclosed that microorganisms, especially bacteria, adhere to surfaces having pendant phosphoryl choline groups than to similar surfaces without such groups present.
The present invention relates to novel biocompatible polymers, the use of these polymers to improve binding of mucopolysaccharides to substrates to improve their hemocompatibility and compositions containing mucopolysaccharides and the polymer.
In WO-A-93/01221 we describe various polymers and their use to coat surfaces to improve their biocompatibility. The polymers include zwitterionic groups ZO and pendant groups which are capable of providing stable surface binding of the polymer to underlying substrate surfaces. The binding may be by provision of pendant hydrophobic groups which physisorb onto hydrophobic substrates, by counterionic attraction between pendant ionic groups on the polymer and oppositely charged groups at the substrate surface, by providing covalent attachment between coreactive pendant groups on the polymer and groups at the substrate surface or by crosslinking the polymer after coating. Post coating crosslinking may also be used to improve the stability of a polymer which is physisorbed, covalently bonded or counterionically bonded to the surface. The polymers have good hemocompatibility as indicated by the low platelet adhesion values reported in that specification.
It has also been shown that zwitterionic groups at substrate surfaces, for instance of contact lenses, show lower rates of deposition of proteins and lipids from biological liquids such as tear film. In WO-A-92/07885, reduced levels of protein deposition are described for contact lenses formed from a hydrogel of a crosslinked copolymer of copolymerisable zwitterionic monomer and non ionic comonomer.
In WO-A-93/21970 it is disclosed that microorganisms, especially bacteria, adhere to surfaces having pendant phosphoryl choline groups than to similar surfaces without such groups present.
Another way of reducing the thrombogenicity of surfaces has involved attachment or adsorption of anti-thrombogenic active compounds to substrate surfaces. For instance heparin may be attached through covalent or counterionic bonding to surfaces. In US-A-3,634,123 the binding of heparin to a surface was increased by incorporation of cationic surfactant. A related process is described in EP-A-0350161, in which a surface is first coated with a cationic surfactant and subsequently with heparin. In EP-A-0086187 the surface is first coated with a cationic polymer and subsequently with heparin. In JP-A-53/137268 a cross-linked acrylic copolymer of a cationic monomer and a polyethyleneglycol monomer is blended with polyurethane and made into tubing which can be coated with heparin. In EP-A-0086186 heparin is attached to an underlying surface through a covalent bond via the end carbohydrate unit. In US-A-5,342,621, a complex is formed of heparin with phosphatidyl choline and admixed with a polymer of caprolactone or L-lactic acid (both substantially unchanged overall) and subsequently used to coat medical devices.
The present inventors have discovered that the performance of heparin coated devices which are commercially available, for instance as components of extra corporeal devices, deteriorates after short periods of use, for instance half an hour. It is not known whether this is due to the heparin being removed from the surface or due to the surface becoming fouled by components of blood or other biological liquid in contact with the surface during use such that the heparin is masked. The present invention seeks to provide a substrate surface which is hemocompatible and retains its hemocompatible properties over longer term in use.
Generally patients who are undergoing complex operations requiring that their blood be directed through extra corporeal circuitry, require administration of heparin into the circulation to prevent the blood clotting.
The present inventors have discovered that the performance of heparin coated devices which are commercially available, for instance as components of extra corporeal devices, deteriorates after short periods of use, for instance half an hour. It is not known whether this is due to the heparin being removed from the surface or due to the surface becoming fouled by components of blood or other biological liquid in contact with the surface during use such that the heparin is masked. The present invention seeks to provide a substrate surface which is hemocompatible and retains its hemocompatible properties over longer term in use.
Generally patients who are undergoing complex operations requiring that their blood be directed through extra corporeal circuitry, require administration of heparin into the circulation to prevent the blood clotting.
Subsequently the heparin has to be neutralised or removed from the blood stream. In order to remove heparin from the circulation without administering a further active compound to neutralise the heparin, it has been suggested to immobilise protamine, a cationic polypeptide used to neutralise heparin, at the surfaces of a filter used in an extra corporeal blood circuit, to scavenge heparin from a patient systemically heparinised.
In US-A-3,861,948 pressure sensitive adhesives are copolymers of ionic monomer and alkylacrylate monomers.
The ionic monomers may be cationic and/or zwitterionic.
Zwitterionic monomers are sulpho or carboxy-betaines, and are used in combination with permanent cationic monomers and monomers with glycidyl groups which can be reacted after coating to cross-link the polymer.
A new terpolymer according to the invention has an overall cationic charge and is formed from ethylenically unsaturated monomers including a) a zwitterionic monomer of the formula I
YBX I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally include one or more fluorine substituents;
X is an organic group having a zwitterionic moiety;
and Y is an ethylenically unsaturated polymerisable group;
b) a cationic monomer of the formula II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally includes one or more fluorine substituents;
In US-A-3,861,948 pressure sensitive adhesives are copolymers of ionic monomer and alkylacrylate monomers.
The ionic monomers may be cationic and/or zwitterionic.
Zwitterionic monomers are sulpho or carboxy-betaines, and are used in combination with permanent cationic monomers and monomers with glycidyl groups which can be reacted after coating to cross-link the polymer.
A new terpolymer according to the invention has an overall cationic charge and is formed from ethylenically unsaturated monomers including a) a zwitterionic monomer of the formula I
YBX I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally include one or more fluorine substituents;
X is an organic group having a zwitterionic moiety;
and Y is an ethylenically unsaturated polymerisable group;
b) a cationic monomer of the formula II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally includes one or more fluorine substituents;
Y' is an ethylenically unsaturated polymerisable group; and Q is an organic group having a cationic or cationisable moiety; and c) a termonomer of the formula III
YzBzQz III
wherein BZ is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which may optionally include one or more fluorine substituents;
Y' is an ethylenically unsaturated polyrnerisable group; and Qz is an organic group having a hydrophobic group selected from alkyl groups having at least six carbon atoms, fluorine substituted alkyl groups and alkyl groups having at least one siloxane substituent.
The terpolymer may include pendant groups capable of providing covalent bonding at the substrate surface or cross-linking between polymer chains. Such groups are generally introduced by incorporation of additional reactive monomers into the monomer mixture. A termonomer may, for instance, comprise a covalent reactive group which is capable of forming a covalent bond with coreactive groups at the substrate surface. Alternatively the copolymer may be crosslinked after coating by subjecting a polymer having pendant crosslinkable groups to conditions such that crosslinking takes place.
A covalent reactive monomer may have the general formula IV:
wherein B' is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally includes one or more fluorine substituents;
Y' is an ethylenically unsaturated polymerisable group; and 5 Q' is an organic group having a reactive group capable, on imposition of an external stimulus, of reacting with a coreactive group on the surface of a substrate or which is pendant on the polymer.
Reactive groups Q' may also provide crosslinkability on the polymer. For instance such groups may react with each other or may react with different coreactive groups as pendant groups on the copolymer, for instance amine or, more usually, hydroxyl groups. Examples of reactive groups capable of crosslinking with such pendant groups or of reacting to provide covalent binding to a surface, an aidehyde group or a silane or siloxane group containing one or more reactive substituents such as halogen, for example chlorine, or alkoxy, generally containing from 1 to 4 carbon atoms, for example methoxy or ethoxy, or, more preferably, Q' is a hydroxyl, amino, carboxyl, epoxy, -CHOHCH,Hal, (in which Hal is a halogen atom such as chlorine, bromine or iodine) succinimido, tosylate, triflate, imidazole carbonyl-amino or optionally substituted triazine group. A preferred example of a reactive group is a trimethoxysilyl group which reacts either with other similar groups or with hydroxyl groups on the terpolymer or a substrate.
Preferred reactive comonomers IV which are used to crosslink the comonomer, rather than provide covalent binding to the surface, are those Q' contains a crosslinkable cinnamyl, epoxy, -CHOHCHZHal (in which Hal is a halogen atom), methylol, reactive silyl, an ethylenically unsaturated crosslinkable group, such as an acetylenic, diacetylenic, vinylic or divinylic group, or an acetoacetoxy or chloroalkyl sulfone, preferably chloroethyl sulphone, group. For optimum cross-linking a monomer including a reactive silyl group is used in combination with a further monomer including a hydroxyl group.
Such polymers may include a diluent monomer, for instance of the types described below.
It is believed that quater polymers of a zwitterionic monomer of formula I above, a cationic monomer of formula II above, a reactive monomer of formula IV in which Q' is a trialkoxysilyl group and a quater monomer of the formula IV in which Q' is a hydroxyl group are new and are claimed herein.
In each of the monomers I to IV the ethylenically unsaturated group is preferably selected from R
CHI=C-C-A- , O ~'' CHZ=C (R) -CHz-O-, CH,=C (R) -CHZOC (O) -, CH~=C (R) OC (O) -, CHI=C (R) O-, and CH,=C (R) CHZOC (O) N (R') -wherein:
R is hydrogen or a C1-Cq alkyl group;
A is -O- or -NR'- where R' is hydrogen or a C,-Ca alkyl group or R' is -B-X, B'Q1, BZQZ or B'Q' where B, B', B', B', Ql, Qz and Q' and X are as defined above in the respective formula I to IV and K is a group -(CHZ)POC(O)-, -(CHz)pC(O)O-, -(CHZ)POC(O)O-, -(CHZ)PNRZ-, -(CHZ)PNRZC(O)-, -(CHz)PC(O)NRz-, -(CH~)PNRZC(O)O-, -(CHz)pOC(O)NR'-, -(CHZ)PNRZC(0)NRZ-, (in which the groups R' are the same or different) -(CHZ)PO-, -(CHZ)PSO, -, or, optionally in combination with B, a valence bond and p is from 1 to 12 and RZ is hydrogen or a C~-C, alkyl group.
Preferably the ethylenically unsaturated groups of all monomers copolymerised together are either the acrylate type or are the styrene type, and, most preferably each has the same formula. Preferably the groups A of acrylate type ethylenically unsaturated groups of the zwitterionic, cationic and termonomer are the same and are most preferably all -O-.
The zwitterionic group X preferably has a phosphate ester group as the anion or the thioester analogue or amide analogue or a phosphonate. The cationic moiety is preferably a quaternary ammonium group, but may be a sulphonium or phosphonium group. Preferably the cationic group is at the end of the group X distant from the group B.
Preferably X is a group of formula O
-Xi _ IP-Xz _W. ~ VI ) I
O-in which the moieties X' and Xz, which are the same or different, are -O-, -S-, -NH- or a valence bond, preferably -O-, and W' is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C,_,2-alkylene group.
Preferably W contains as cationic group an ammonium group, more preferably a quaternary ammonium group.
The group W' may for example be a group of formula -W1-N.Rz33 ~ -W:-P.Rz3a3 ~ -Wl-S.Rzsaz or -W'-Het' iri which W' is alkylene of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W' optionally contains one or more fluorine substituents and/or one or more functional groups; and either the groups Rz' are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R-' together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R'' together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups Rz' is substituted by a hydrophilic functional group, and the groups RZ'a are the same or different and each is RZ' or a group ORZ', where RZ' is as defined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.
Preferably W' is a straight-chain alkylene group, most preferably 1,2-ethylene.
Preferred groups X of the formula VI are groups of formula VA.
The groups of formula (VA) are:
O
2 0 -O-P-O ( CHZ ) e-N" ( R12 ) 3 ( VA ) Oa where the groups R1z are the same or different and each is hydrogen or C1_4 alkyl, and a is from 1 to 4.
Preferably the groups R'2 are the same. It is also preferable that at least one of the groups R'' is methyl, and more preferable that the groups R'2 are all methyl.
Preferably a is 2 or 3, more preferably 2.
When X is a group of formula (VA} preferably B is a group of formula -(CR1'z)- or -(CR132)2-, eg. -(CH,)- or -(CH,CH~)-.
Preferably the zwitterionic monomer has the general formula VI
R O O
CHZ = C- (C-A-B-O- IP-O- ( CHz ) eNi ( R3 ) 3 VI
O-wherein R, A and B are defined above, the groups R' are the same or different and each is . hydrogen C1_, alkyl, aryl, alkaryl, aralkyl, or two or three of the groups RI with the nitrogen atom to which they are attached form a saturated or unsaturated hetero cyclic ring, and a is 1 to 6, preferably 2 to 4.
A cationisable moiety in the group Q' is generally a group which can easily be protonated to render it cationic, for instance which is protonated in aqueous environments at pH7.
The group Q' of the cationic monomer is preferably a group N'R53, P'R5~ or S'Rsz in which the groups RS are the same or different and are each hydrogen, C,_a-alkyl or aryl (preferably phenyl) or two of the groups RS together with the heteroatom to which they are attached from a saturated or unsaturated heterocyclic ring containing from 5 to 7 atoms. Preferably the group Q1 is permanently cationic, that is each RS is other than hydrogen. Preferably Q' is N'R5, in which each R5 is C,_4-alkyl, preferably methyl.
Monomer formulations suitable for forming the novel terpolymers and novel quater polymers are claimed herein.
Liquid compositions containing the terpolymers and quater polymers and a solvent are claimed herein as are processes in which the liquid composition is coated onto a surface and the solvent is removed to leave a coating on the surface.
By incorporating pendant groups to provide stable binding on the surface, the terpolymers and quater polymers can be stably bound to many types of underlying surface, for subsequent provision of a coated substrate for receiving heparin.
In a new application of the novel terpolymer or quater polymer according to the invention a substrate having a coating of the polymer is contacted with a solution having suspended or dissolved therein an anionically charged mucopolysaccharide.
The anionically charged mucopolysaccharide may be heparin or a similar anti-thrombogenic compound such as hirudin or chondroitin sulphate, or may be alginate or hyaluronic acid. The provision of cationic pendant groups 5 at the substrate surface on the coating provides a charged entity, having the opposite charge to that of the mucopolysaccharide, enabling the mucopolysaccharide to become counterionically bonded to the surface in the contacting step. The zwitterionic groups seem to minimise 10 adsorption of other components from blood or biological fluids subsequently contacted with the coated surface, thereby preventing fouling of the surface which would mask the mucopolysaccharide's effect.
The mucopolysaccharide coating may be carried out as the second step of a two step process. In the first step a substrate is coated with a liquid composition containing the polymer suspended or dissolved in a solvent, followed by removal of the solvent prior to the mucopolysaccharide coating step.
Alternatively the pendant groups Q' and/or Q' may provide compatibility with other polymers when blended, for instance by solid or liquid blending techniques. Thus pendant hydrophobic groups may interact with hydrophobic blended copolymers whilst reactive groups may be crosslinked, for instance during reactive blending processes or after blending has taken place. Such blends can subsequently be used to form shaped articles which may be coated with heparin in a post shaping step. The novel polymers themselves may have satisfactory properties such that they may be useful to form components of devices which can be treated with heparin to improve their anti-thrombogenicity.
The novel terpolymer may be blended with heparin in a pre-blending step and the complex subsequently used to form coatings or be used in a blend with other polymers having desirable mechanical characteristics. A blend may, for instance, be made by dispersing both components in a solvent in which they are both compatible. Alternatively each component is dissolved or dispersed in a solvent which is suitable for the respective component and the two liquid compositions mixed. Other components may be included to stabilise the mixture. Such pre-blended heparin/polymer complexes are primarily of use as coating components, that is for forming deposits from liquid coating compositions onto underlying substrate surfaces.
A complex formed by crosslinking anionic mucopolysaccharides by the novel terpolymer forms a further aspect of the present invention. The cationic groups of the polymer provide intermolecular ionic crosslinking with the mucopolysaccharide molecules.
The zwitterionic terpolymer or quater polymer and the anionic mucopolysaccharide are generally used in ratios of equivalent ionic groups in the range 1:10 to 10:1, preferably about 1:2 to 2:1, probably about 1:1. The use of such ratios allows the formation of a crosslinked mucopolysaccharide which may have suitable characteristics such that a gel, comprising a liquid component in which the crosslinked mucopolysaccharide is swellable but not soluble. Such gels may be used for instance as wound dressings, microbial culture media, drug delivery systems, etc. The dry crosslinked materials may be used as absorbent materials for absorbing aqueous or organic solvent based liquids.
The novel terpolymers or quaterpolymers may also be used in ion exchange resins or in other separation processes. For instance particles or membranes of or coated by the terpolymer or quater polymer may be used to remove anionic components from liquids in which they are suspended or dissolved. This use of the zwitterionic/cationic resins minimises adsorption from such a liquid of other components, by reducing the extent to which such other components adhere to the resin through the activity of the zwitterionic groups. This minimises fouling of the resin by components other than those intended to be ion exchanged. This may also be desirable where the resin is used to remove anionic components from fluids intended to be subsequently introduced or reintroduced into a patient's body. In this aspect, blood may be circulated through a bed of particles of such a resin or through a membrane or other filter formed of such a resin to remove heparin from the circulation in a patient subjected to heparin treatment for surgery or other reasons.
We have found that the terpolymers and quater polymers described in the examples herein can be used successfully to coat various substrates including polyesters, polycarbonates polypropylene, polyvinyl chloride and steel and filters may include coated surfaces of any of these materials.
Instead of passing anticoagulant-treated blood through an extra corporeal filter, heparin (or other anticoagulant) scavenging may be carried out by implanting, permanently or temporarily, a device into the body in the circulation, which can remove anticoagulant which has been administered systemically. Thus the terpolymer or quater polymer may be coated onto the surface of a vascular stent introduced into a blood vessel of a patient. In this embodiment the device may act as a reservoir, formed in situ, of active ingredient which may be released slowly into the circulation over an extended period of time. Alternatively a device may be preloaded with counterionically charged mucopolysaccharide prior to implantation, to act as a slow release drug delivery system.
The proportions of zwitterionic and cationic pendant groups in the novel polymers depends upon the desired end use. Where high levels of mucopolysaccharide are to be scavenged from a fluid composition and/or it is desired for a high density of anionic mucopolysaccharide to be deposited onto a surface for subsequent use, then the amount of cationic pendant group should be relatively high as compared to the levels of zwitterionic groups. However where lower levels of mucopolysaccharide are required to be adsorbed to achieve anti-thrombogenic performance, whilst minimising deposition of protein and lipid components and platelets forms an important characteristic of the surfaces, then high levels of zwitterionic pendant groups are likely to be desirable. The relative ratios (equivalents) is in the range 1:100 to 100:1 (zwitterionic to ionic) preferably 1:10 to 10:1, more preferably 1:2 to 20:1.
The total molar proportion of monomer of the formula III or IV in the polymer may be in the range 0.1 to 750.
The polymers may include diluent comonomer. Such diluent comonomer may be used in quantities up to 90 mol%, usually less than 50 mol%. Copolymerisable nonionic monomers may be used such as C1_24 alkyl(meth}acrylates, -(meth) acrylamides, and hydroxy C1_z4alkyl (meth) acrylates and (meth)acrylamides.
The terpolymers may include anionic pendant groups, to provide intermolecular crosslinking by counterionic bonding with cationic groups. In such cases, the equivalent level of anionic groups is lower than that of cationic groups in order that the polymer has an overall cationic charge.
Anionic copolymerisable monomers may be used, for instance in which the anionic group is derived from carboxylic, sulphonic or phosphonic acid.
It has been found that the binding of heparin and the terpolymer or quaterpolymer to a surface provides a coated substrate in which the heparin appears to be in a condition such that good anti-thrombogenic properties are exhibited.
Furthermore the coating is very stable and resistant to fouling during use such that the heparin conferred properties are retained even after substantial periods of use. Thus the performance is found to be greatly improved as compared to normal heparin treated surfaces. The binding of heparin to pretreated surfaces which have pendant cationic and zwitterionic groups is achieved merely by contacting the surface with heparin in solution, whereby heparin becomes bound to the cationic groups via counterionic bonding.
The following examples illustrates the inventions.
Performance Tests Heparin Activity Loading of samples with heparin 1. Filter strips.
Samples were incubated with 5 ml of a solution of heparin in PBS (usually 50 U/ml. In other experiments, a heparin concentration of 4 or 200 U/ml in saline produced the same heparin surface activity on the cationic polymer) for 30 min on a test tube shaker at room temperature.
After 30 min, the samples were rinsed for 10 sec on both sides first with PBS then with deionized water. The samples were dried on tissue paper and in air and stored at room temperature.
2. Whole filters.
Arterial filters were filled with 100 ml of a heparin solution in PBS (50 U/ml) and inlet/outlet sides were closed. The filter was rotated for 30 min, ensuring that all parts of the device were in contact with the heparin loading solution. The filter was then drained and filled/drained 3 times with PBS and then filled/drained 3 times with deionized water. The filter was dried by a stream of air and stored at room temperature.
separation of samples for heparin test Heparin loaded filter strips (dip-coated or removed from whole arterial filters) were usually incubated for 5 hrs at 37°C in PBS/BSA 1°s/NaN3 0.1% to remove unstable bound heparin. The samples were then rinsed with PBS and deionized water as described and dried in air. Samples of 0.2-0.4 x 0.4 cm were cut out and tested as described below.
Heparin test A chromogenic assay (Heparin CRS106, Sigma). The "Semi-Micro Method" described in the manual was used.
Heparin loaded coated samples were placed in polystyrene test tubes. The tubes were placed into a 37°C water bath (5 tubes). 200 ~,1 of bovine factor Xa was added and the tubes were shaken. Following 1 min agitation, 200 ~,1 5 factor Xa substrate was added to the tubes and they were agitated for 5 min. 200 ~,1 acetic acid (>90%) was added to the tubes and the tubes were shaken. 200 ~1 of the solution was removed from the tubes and added to the well of a microplate (2 ~wells/sample) and measured at 405 nm 10 against wells containing 200 ~,1 of PBS. Previous results had shown that PBS gave the same absorbance reading as a reagent blank. The heparin activity was calculated with the use of a standard curve prepared with soluble heparin.
Platelet adhesion 15 Heparin loaded and heparin free samples were incubated with human blood (citrate or heparin as anticoagulant) for 2-3 hrs and the degree of platelet adhesion was determined by scanning electron microscopy.
Fibrinocren absorbance Samples of heparin loaded or heparin-free coated material were incubated with human plasma for l0 min, washed with PBS/BSA 1%, then incubated for 30 min with an anti-human fibrinogen antibody conjugated to horse radish peroxidase (Dako Code No. A080). The samples were washed and bound antibody was determined by incubating the samples with a substrate for peroxidase (0-phenylenediamine dihydrochloride, 0.4 mg/ml) and a phosphate citrate buffer with urea hydrogen peroxide (Sigma P-9305). After 10 min the absorbance at 450 nm was measured against a reagent blank.
Perfusion with bovine blood Two arterial filters (a control filter and a coated heparin loaded filter or a coated non-heparin loaded filter) were perfused in parallel for 6 hrs with bovine blood (3.5 L/min) at reduced heparin concentrations and macroscopic blood clots were detected visually and photographs were taken.
YzBzQz III
wherein BZ is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which may optionally include one or more fluorine substituents;
Y' is an ethylenically unsaturated polyrnerisable group; and Qz is an organic group having a hydrophobic group selected from alkyl groups having at least six carbon atoms, fluorine substituted alkyl groups and alkyl groups having at least one siloxane substituent.
The terpolymer may include pendant groups capable of providing covalent bonding at the substrate surface or cross-linking between polymer chains. Such groups are generally introduced by incorporation of additional reactive monomers into the monomer mixture. A termonomer may, for instance, comprise a covalent reactive group which is capable of forming a covalent bond with coreactive groups at the substrate surface. Alternatively the copolymer may be crosslinked after coating by subjecting a polymer having pendant crosslinkable groups to conditions such that crosslinking takes place.
A covalent reactive monomer may have the general formula IV:
wherein B' is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally includes one or more fluorine substituents;
Y' is an ethylenically unsaturated polymerisable group; and 5 Q' is an organic group having a reactive group capable, on imposition of an external stimulus, of reacting with a coreactive group on the surface of a substrate or which is pendant on the polymer.
Reactive groups Q' may also provide crosslinkability on the polymer. For instance such groups may react with each other or may react with different coreactive groups as pendant groups on the copolymer, for instance amine or, more usually, hydroxyl groups. Examples of reactive groups capable of crosslinking with such pendant groups or of reacting to provide covalent binding to a surface, an aidehyde group or a silane or siloxane group containing one or more reactive substituents such as halogen, for example chlorine, or alkoxy, generally containing from 1 to 4 carbon atoms, for example methoxy or ethoxy, or, more preferably, Q' is a hydroxyl, amino, carboxyl, epoxy, -CHOHCH,Hal, (in which Hal is a halogen atom such as chlorine, bromine or iodine) succinimido, tosylate, triflate, imidazole carbonyl-amino or optionally substituted triazine group. A preferred example of a reactive group is a trimethoxysilyl group which reacts either with other similar groups or with hydroxyl groups on the terpolymer or a substrate.
Preferred reactive comonomers IV which are used to crosslink the comonomer, rather than provide covalent binding to the surface, are those Q' contains a crosslinkable cinnamyl, epoxy, -CHOHCHZHal (in which Hal is a halogen atom), methylol, reactive silyl, an ethylenically unsaturated crosslinkable group, such as an acetylenic, diacetylenic, vinylic or divinylic group, or an acetoacetoxy or chloroalkyl sulfone, preferably chloroethyl sulphone, group. For optimum cross-linking a monomer including a reactive silyl group is used in combination with a further monomer including a hydroxyl group.
Such polymers may include a diluent monomer, for instance of the types described below.
It is believed that quater polymers of a zwitterionic monomer of formula I above, a cationic monomer of formula II above, a reactive monomer of formula IV in which Q' is a trialkoxysilyl group and a quater monomer of the formula IV in which Q' is a hydroxyl group are new and are claimed herein.
In each of the monomers I to IV the ethylenically unsaturated group is preferably selected from R
CHI=C-C-A- , O ~'' CHZ=C (R) -CHz-O-, CH,=C (R) -CHZOC (O) -, CH~=C (R) OC (O) -, CHI=C (R) O-, and CH,=C (R) CHZOC (O) N (R') -wherein:
R is hydrogen or a C1-Cq alkyl group;
A is -O- or -NR'- where R' is hydrogen or a C,-Ca alkyl group or R' is -B-X, B'Q1, BZQZ or B'Q' where B, B', B', B', Ql, Qz and Q' and X are as defined above in the respective formula I to IV and K is a group -(CHZ)POC(O)-, -(CHz)pC(O)O-, -(CHZ)POC(O)O-, -(CHZ)PNRZ-, -(CHZ)PNRZC(O)-, -(CHz)PC(O)NRz-, -(CH~)PNRZC(O)O-, -(CHz)pOC(O)NR'-, -(CHZ)PNRZC(0)NRZ-, (in which the groups R' are the same or different) -(CHZ)PO-, -(CHZ)PSO, -, or, optionally in combination with B, a valence bond and p is from 1 to 12 and RZ is hydrogen or a C~-C, alkyl group.
Preferably the ethylenically unsaturated groups of all monomers copolymerised together are either the acrylate type or are the styrene type, and, most preferably each has the same formula. Preferably the groups A of acrylate type ethylenically unsaturated groups of the zwitterionic, cationic and termonomer are the same and are most preferably all -O-.
The zwitterionic group X preferably has a phosphate ester group as the anion or the thioester analogue or amide analogue or a phosphonate. The cationic moiety is preferably a quaternary ammonium group, but may be a sulphonium or phosphonium group. Preferably the cationic group is at the end of the group X distant from the group B.
Preferably X is a group of formula O
-Xi _ IP-Xz _W. ~ VI ) I
O-in which the moieties X' and Xz, which are the same or different, are -O-, -S-, -NH- or a valence bond, preferably -O-, and W' is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C,_,2-alkylene group.
Preferably W contains as cationic group an ammonium group, more preferably a quaternary ammonium group.
The group W' may for example be a group of formula -W1-N.Rz33 ~ -W:-P.Rz3a3 ~ -Wl-S.Rzsaz or -W'-Het' iri which W' is alkylene of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W' optionally contains one or more fluorine substituents and/or one or more functional groups; and either the groups Rz' are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R-' together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R'' together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups Rz' is substituted by a hydrophilic functional group, and the groups RZ'a are the same or different and each is RZ' or a group ORZ', where RZ' is as defined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.
Preferably W' is a straight-chain alkylene group, most preferably 1,2-ethylene.
Preferred groups X of the formula VI are groups of formula VA.
The groups of formula (VA) are:
O
2 0 -O-P-O ( CHZ ) e-N" ( R12 ) 3 ( VA ) Oa where the groups R1z are the same or different and each is hydrogen or C1_4 alkyl, and a is from 1 to 4.
Preferably the groups R'2 are the same. It is also preferable that at least one of the groups R'' is methyl, and more preferable that the groups R'2 are all methyl.
Preferably a is 2 or 3, more preferably 2.
When X is a group of formula (VA} preferably B is a group of formula -(CR1'z)- or -(CR132)2-, eg. -(CH,)- or -(CH,CH~)-.
Preferably the zwitterionic monomer has the general formula VI
R O O
CHZ = C- (C-A-B-O- IP-O- ( CHz ) eNi ( R3 ) 3 VI
O-wherein R, A and B are defined above, the groups R' are the same or different and each is . hydrogen C1_, alkyl, aryl, alkaryl, aralkyl, or two or three of the groups RI with the nitrogen atom to which they are attached form a saturated or unsaturated hetero cyclic ring, and a is 1 to 6, preferably 2 to 4.
A cationisable moiety in the group Q' is generally a group which can easily be protonated to render it cationic, for instance which is protonated in aqueous environments at pH7.
The group Q' of the cationic monomer is preferably a group N'R53, P'R5~ or S'Rsz in which the groups RS are the same or different and are each hydrogen, C,_a-alkyl or aryl (preferably phenyl) or two of the groups RS together with the heteroatom to which they are attached from a saturated or unsaturated heterocyclic ring containing from 5 to 7 atoms. Preferably the group Q1 is permanently cationic, that is each RS is other than hydrogen. Preferably Q' is N'R5, in which each R5 is C,_4-alkyl, preferably methyl.
Monomer formulations suitable for forming the novel terpolymers and novel quater polymers are claimed herein.
Liquid compositions containing the terpolymers and quater polymers and a solvent are claimed herein as are processes in which the liquid composition is coated onto a surface and the solvent is removed to leave a coating on the surface.
By incorporating pendant groups to provide stable binding on the surface, the terpolymers and quater polymers can be stably bound to many types of underlying surface, for subsequent provision of a coated substrate for receiving heparin.
In a new application of the novel terpolymer or quater polymer according to the invention a substrate having a coating of the polymer is contacted with a solution having suspended or dissolved therein an anionically charged mucopolysaccharide.
The anionically charged mucopolysaccharide may be heparin or a similar anti-thrombogenic compound such as hirudin or chondroitin sulphate, or may be alginate or hyaluronic acid. The provision of cationic pendant groups 5 at the substrate surface on the coating provides a charged entity, having the opposite charge to that of the mucopolysaccharide, enabling the mucopolysaccharide to become counterionically bonded to the surface in the contacting step. The zwitterionic groups seem to minimise 10 adsorption of other components from blood or biological fluids subsequently contacted with the coated surface, thereby preventing fouling of the surface which would mask the mucopolysaccharide's effect.
The mucopolysaccharide coating may be carried out as the second step of a two step process. In the first step a substrate is coated with a liquid composition containing the polymer suspended or dissolved in a solvent, followed by removal of the solvent prior to the mucopolysaccharide coating step.
Alternatively the pendant groups Q' and/or Q' may provide compatibility with other polymers when blended, for instance by solid or liquid blending techniques. Thus pendant hydrophobic groups may interact with hydrophobic blended copolymers whilst reactive groups may be crosslinked, for instance during reactive blending processes or after blending has taken place. Such blends can subsequently be used to form shaped articles which may be coated with heparin in a post shaping step. The novel polymers themselves may have satisfactory properties such that they may be useful to form components of devices which can be treated with heparin to improve their anti-thrombogenicity.
The novel terpolymer may be blended with heparin in a pre-blending step and the complex subsequently used to form coatings or be used in a blend with other polymers having desirable mechanical characteristics. A blend may, for instance, be made by dispersing both components in a solvent in which they are both compatible. Alternatively each component is dissolved or dispersed in a solvent which is suitable for the respective component and the two liquid compositions mixed. Other components may be included to stabilise the mixture. Such pre-blended heparin/polymer complexes are primarily of use as coating components, that is for forming deposits from liquid coating compositions onto underlying substrate surfaces.
A complex formed by crosslinking anionic mucopolysaccharides by the novel terpolymer forms a further aspect of the present invention. The cationic groups of the polymer provide intermolecular ionic crosslinking with the mucopolysaccharide molecules.
The zwitterionic terpolymer or quater polymer and the anionic mucopolysaccharide are generally used in ratios of equivalent ionic groups in the range 1:10 to 10:1, preferably about 1:2 to 2:1, probably about 1:1. The use of such ratios allows the formation of a crosslinked mucopolysaccharide which may have suitable characteristics such that a gel, comprising a liquid component in which the crosslinked mucopolysaccharide is swellable but not soluble. Such gels may be used for instance as wound dressings, microbial culture media, drug delivery systems, etc. The dry crosslinked materials may be used as absorbent materials for absorbing aqueous or organic solvent based liquids.
The novel terpolymers or quaterpolymers may also be used in ion exchange resins or in other separation processes. For instance particles or membranes of or coated by the terpolymer or quater polymer may be used to remove anionic components from liquids in which they are suspended or dissolved. This use of the zwitterionic/cationic resins minimises adsorption from such a liquid of other components, by reducing the extent to which such other components adhere to the resin through the activity of the zwitterionic groups. This minimises fouling of the resin by components other than those intended to be ion exchanged. This may also be desirable where the resin is used to remove anionic components from fluids intended to be subsequently introduced or reintroduced into a patient's body. In this aspect, blood may be circulated through a bed of particles of such a resin or through a membrane or other filter formed of such a resin to remove heparin from the circulation in a patient subjected to heparin treatment for surgery or other reasons.
We have found that the terpolymers and quater polymers described in the examples herein can be used successfully to coat various substrates including polyesters, polycarbonates polypropylene, polyvinyl chloride and steel and filters may include coated surfaces of any of these materials.
Instead of passing anticoagulant-treated blood through an extra corporeal filter, heparin (or other anticoagulant) scavenging may be carried out by implanting, permanently or temporarily, a device into the body in the circulation, which can remove anticoagulant which has been administered systemically. Thus the terpolymer or quater polymer may be coated onto the surface of a vascular stent introduced into a blood vessel of a patient. In this embodiment the device may act as a reservoir, formed in situ, of active ingredient which may be released slowly into the circulation over an extended period of time. Alternatively a device may be preloaded with counterionically charged mucopolysaccharide prior to implantation, to act as a slow release drug delivery system.
The proportions of zwitterionic and cationic pendant groups in the novel polymers depends upon the desired end use. Where high levels of mucopolysaccharide are to be scavenged from a fluid composition and/or it is desired for a high density of anionic mucopolysaccharide to be deposited onto a surface for subsequent use, then the amount of cationic pendant group should be relatively high as compared to the levels of zwitterionic groups. However where lower levels of mucopolysaccharide are required to be adsorbed to achieve anti-thrombogenic performance, whilst minimising deposition of protein and lipid components and platelets forms an important characteristic of the surfaces, then high levels of zwitterionic pendant groups are likely to be desirable. The relative ratios (equivalents) is in the range 1:100 to 100:1 (zwitterionic to ionic) preferably 1:10 to 10:1, more preferably 1:2 to 20:1.
The total molar proportion of monomer of the formula III or IV in the polymer may be in the range 0.1 to 750.
The polymers may include diluent comonomer. Such diluent comonomer may be used in quantities up to 90 mol%, usually less than 50 mol%. Copolymerisable nonionic monomers may be used such as C1_24 alkyl(meth}acrylates, -(meth) acrylamides, and hydroxy C1_z4alkyl (meth) acrylates and (meth)acrylamides.
The terpolymers may include anionic pendant groups, to provide intermolecular crosslinking by counterionic bonding with cationic groups. In such cases, the equivalent level of anionic groups is lower than that of cationic groups in order that the polymer has an overall cationic charge.
Anionic copolymerisable monomers may be used, for instance in which the anionic group is derived from carboxylic, sulphonic or phosphonic acid.
It has been found that the binding of heparin and the terpolymer or quaterpolymer to a surface provides a coated substrate in which the heparin appears to be in a condition such that good anti-thrombogenic properties are exhibited.
Furthermore the coating is very stable and resistant to fouling during use such that the heparin conferred properties are retained even after substantial periods of use. Thus the performance is found to be greatly improved as compared to normal heparin treated surfaces. The binding of heparin to pretreated surfaces which have pendant cationic and zwitterionic groups is achieved merely by contacting the surface with heparin in solution, whereby heparin becomes bound to the cationic groups via counterionic bonding.
The following examples illustrates the inventions.
Performance Tests Heparin Activity Loading of samples with heparin 1. Filter strips.
Samples were incubated with 5 ml of a solution of heparin in PBS (usually 50 U/ml. In other experiments, a heparin concentration of 4 or 200 U/ml in saline produced the same heparin surface activity on the cationic polymer) for 30 min on a test tube shaker at room temperature.
After 30 min, the samples were rinsed for 10 sec on both sides first with PBS then with deionized water. The samples were dried on tissue paper and in air and stored at room temperature.
2. Whole filters.
Arterial filters were filled with 100 ml of a heparin solution in PBS (50 U/ml) and inlet/outlet sides were closed. The filter was rotated for 30 min, ensuring that all parts of the device were in contact with the heparin loading solution. The filter was then drained and filled/drained 3 times with PBS and then filled/drained 3 times with deionized water. The filter was dried by a stream of air and stored at room temperature.
separation of samples for heparin test Heparin loaded filter strips (dip-coated or removed from whole arterial filters) were usually incubated for 5 hrs at 37°C in PBS/BSA 1°s/NaN3 0.1% to remove unstable bound heparin. The samples were then rinsed with PBS and deionized water as described and dried in air. Samples of 0.2-0.4 x 0.4 cm were cut out and tested as described below.
Heparin test A chromogenic assay (Heparin CRS106, Sigma). The "Semi-Micro Method" described in the manual was used.
Heparin loaded coated samples were placed in polystyrene test tubes. The tubes were placed into a 37°C water bath (5 tubes). 200 ~,1 of bovine factor Xa was added and the tubes were shaken. Following 1 min agitation, 200 ~,1 5 factor Xa substrate was added to the tubes and they were agitated for 5 min. 200 ~,1 acetic acid (>90%) was added to the tubes and the tubes were shaken. 200 ~1 of the solution was removed from the tubes and added to the well of a microplate (2 ~wells/sample) and measured at 405 nm 10 against wells containing 200 ~,1 of PBS. Previous results had shown that PBS gave the same absorbance reading as a reagent blank. The heparin activity was calculated with the use of a standard curve prepared with soluble heparin.
Platelet adhesion 15 Heparin loaded and heparin free samples were incubated with human blood (citrate or heparin as anticoagulant) for 2-3 hrs and the degree of platelet adhesion was determined by scanning electron microscopy.
Fibrinocren absorbance Samples of heparin loaded or heparin-free coated material were incubated with human plasma for l0 min, washed with PBS/BSA 1%, then incubated for 30 min with an anti-human fibrinogen antibody conjugated to horse radish peroxidase (Dako Code No. A080). The samples were washed and bound antibody was determined by incubating the samples with a substrate for peroxidase (0-phenylenediamine dihydrochloride, 0.4 mg/ml) and a phosphate citrate buffer with urea hydrogen peroxide (Sigma P-9305). After 10 min the absorbance at 450 nm was measured against a reagent blank.
Perfusion with bovine blood Two arterial filters (a control filter and a coated heparin loaded filter or a coated non-heparin loaded filter) were perfused in parallel for 6 hrs with bovine blood (3.5 L/min) at reduced heparin concentrations and macroscopic blood clots were detected visually and photographs were taken.
Observed Chloride The counter ion in the polymeric system is chloride ion. Quantification of the chloride ion allows the level of cationic methacrylate to be determined.
Procedure Add 0.25 g polymer to 25 ml methanol. Once the material has fully dissolved add 75 ml of distilled water to the polymer/methanol mixture. Adjust the pH of the mixture to fall between 8-9. Add 1.0 ml of potassium chromate (5% in w/v distilled water) by pipette to the flask, and titrated to the first brown/red end-point with standardised 0.01 m silver nitrate solution. Repeat the titration, using 75 ml distilled water, but no polymer sample to obtain a blank reading. The level of cationic methacrylate in the polymer is directly proportional to the chloride ion concentration.
Example 1 Preparation of poly (2(methacryloyloxyethyl)-2' trimethvlammoniuml ethyl phosphate innersalt-co-n-dodecyl methacrylate-co-11 methacryloylundecyl-1-trimet~l ammonium bromide) (40:71:81.
2-(Methacryloyloxyethyl)-2'-(trimethyl ammonium) ethyl phosphate inner salt (2.328, 0.0079 mole), n-dodecyl methacrylate (3.618, 0.0142 mole) and 11 methacryloylundecyl-1-trimethyl ammonium bromide (0.598, 0.0016 mole synthesised according to reference example 1) were dissolved in 43m1 of propan-2-of and 17m1 of ethyl acetate.
This monomer solution Was thoroughly degassed by bubbling dry nitrogen gas (dried over molecular sieve) through it for 30 minutes. The initiator, AIBN (0.013608, 0.02 weight % of solution) was then washed into the solution using 3ml of degassed ethanol. The solution was further degassed for five minutes. Maintaining the solution under a slight positive pressure of nitrogen (equivalent to a few ml of mineral oil in a bubbler) the solution was heated to 62°C and stirred vigorously for around 46 hours.
After this time the reaction mixture was allowed to cool to around 40°C before removing all of the solvent using a rotary evaporator under vacuum and at about 40°C
giving a solid foam.
This foam was then dissolved in 24m1 of dichloromethane and precipitated dropwise into an excess, 200m1, of acetone. The product was collected on a Buchner filter funnel and washed with 3 further 20m1 quantities of acetone. The white solid was dried in a vacuum oven for 16 hours at 40°C and weighed.
The resulting polymer, obtained in 83% yield, was a white solid.
'HNMR (400MHz, d, ppm, CD,OD/CDC13) 4.31(b), 4.21(b), 4.07(b), 3.98(b), 3.72(b),3.37, 3.33, 3.29(s), 3.22, 3.17, 1.95, 1.84(b), 1.67(b), 1.33(s), 1.06(b), 0.93(s), C., NMR
(500MHz, d, ppm, CD30D/CDCI3) 176.37, 66.91, 65.90, 63.68, 60.05, 54.50, 53.37, 45.54, 32.69, 30.44, 30...13, 28.92, 26.93, 23.41, 17.31, 14.56.
Example 2 Preparation of poly f2(methacrvloyloxyethvl)-2' trimethylammonium) ethyl phosphate innersalt-co-n-dodecvl methacrylate-co-cholinemethacrylate Using a similar technique to that used in Example 1, but using choline methacrylate (2-methacryloyloxy-ethyl trimethyl ammonium chloride) in place of 11-methacryloyl undecyl-1-trimethyl ammonium bromide, various polymerisations were carried out. The zwitterionic monomer, lauryl (dodecyl) methacrylate monomer and choline methacrylate were mixed at the molar ratio shown in Table 1 below and AIBN as initiator was used at the level shown in the table. The total weight percent of solids in the polymerisation solution is also reported in the table, since it was varied between examples.
The polymers were recovered by essentially the same method as in claim 1 although including an extra dissolution and precipitation step to remove lower molecular weight polymer.
The polymer product was subjected to chloride ion determination to establish the rate of inclusion of cationic monomer into the product. Also some rough molecular weight determinations were carried out.
Example 3 Preparation of Poly(2-(Methacrylovloxyethyl)-2' (Trimethvlammoniumethyl) Phosphate Inner Salt)-co-(n Dodecyl methacrylate)-co-(2-(Methacrylo,Yloxy) ethyl trimethyl ammonium chloride)-co-(3-Trimethyoxysilylprop3rl methacrvlate) 30:60:6:4 terpolymers 3.1 Monomer Feed Synthesis The zwitterionic monomer (40.68g, 0.138mo1e) and cationic monomer (5.73g,0.0275mo1e) were weighed in a glove box environment dried by P205. Dodecyl methacrylate (69.45g, 0.273mo1e), trimethoxysilyl monomer (4.53g,0.0182mo1e) and a-azo-isobutyronitrile (AIBN) initiator (1.202g, 1%) were weighed in air. A 3 neck reaction flask, fitted with water condenser, nitrogen gas flow and monomer feed tubing, and primed with anhydrous n-propanol (60g) solvent, was immersed in a heated 90°C oil bath. The monomers and initiator were dissolved in 300g of n-propanol solvent and magnetically stirred in a measuring cylinder sealed with parafilm. The reaction mixture was drawn into polypropylene tubing placed inside the measuring cylinder and through silicone tubing via a peristaltic pump to enter the heated reaction vessel in a dropwise process.
A complete transfer to the heated vessel took 2.25 hours.
The reaction was stirred for another hour. A second charge of AIBN initiator (0.12g), dissolved in 3m1 n-propanol, was added and the reaction mixture was stirred for a further 50 min, taking the total reaction time to 4 hours.
Once cooled to room temperature, the reaction mixture was filtered through a sintered glass filter. The solvent was removed at 40°C-50°C by rotary evaporator to give a white foam residue that was later redissolved in 480m1 dichloromethane and 40m1 methanol solvent mixture and dropwise precipitated into 4000m1 acetone. A white solid product settled from the acetone leaving a slightly cloudy supernatant. The product was separated by Buchner flask and 113 Whatman wet strenghtened filter paper, and dried in a room temperature vacuum oven for up to 24 hours prior to a second workup and precipitation in acetone. The product was weighed (82.9g) to provide a 68.9 wt% yield, bottled in a brown glass vial and refridgerated.
Characterisation of Product The polymer requires by weight C 63.08%, H 10.13%, P
3.55%, N 1.93%, Si 0.43% C1 0.81%, found C 58.1%, H 9.98%, P 3.09%, N 1.90%, Si 0.20%, lHnmr (400MHz, ppm, CD,OD:CDC1, 1:1 v:v) 4.34, 4.30, 3.98, 3.72, 3.38, 3.29, 3.22, 1.67, 1.32, 0.92, 0.10. Specific viscosity of l0mg/ml solution in ethanol: chloroform (1:1 v:v) is 0.13. The polymer product was subjected to the chloride ion assay to establish the rate of inclusion of cationic monomer;
required 4.76wt%, found 4.82wt% and 4.94wt%.
3.2 One Pot Synthesis Zwitterionic monomer (4.878, 1.65 x 10-' mole), dodecyl methacrylate (8.llg, 3.19 x 10-Z mole), cationic monomer (0.67g, 0.32 x 10-' mole) and trimethoxy-silyl monomer (0.53g, 0.21 x 10-Z mole) were rinsed into the reaction vessel with 114 ml solvent mixture of 15:85 v/v% MeOH:EtOH.
Anhydrous cationic monomer was predissolved in 3m1 pure MeOH before being rinsed into the reaction vessel. Dodecyl methacrylate monomer was pre-columned through activated basic alumina (Brockmann 1 ca.150 mesh, 50g) before use.
Dry nitrogen gas was bubbled through for 20 minutes to degas the reaction mixture at room temperature before immersing the reaction vessel in an oil bath heated to 67°C. The vessel was heated for 15 minutes prior to AIBN
initiator (0.14g) being rinsed into the reaction mixture with 2m1 solvent mixture. The reaction was magnetically stirred and maintained up a positive pressure nitrogen blanket sufficient to bubble through a mineral oil bubbler.
The reaction time was 39 hours.
Once cooled to room temperature, the reaction mixture appeared clear with a slight haze. The solvent was removed 5 at room temperature by rotary evaporator to give a white foam residue that was later redissolved in 50m1 dichloromethane and added dropwise into vigorously stirred 500m1 acetone. A white solid product settled from the acetone leaving a slightly cloudy supernatant. The product l0 was separated by Buchner flask and 113 Whatman wet strengthened filter paper, and dried in a room temperature vacuum oven far up to 72 hours. The product was weighed to provide a 91 wt% yield, bottled in a glass jar and ref rigerated .
15 Characterisation The polymer requires by weight C 62.93%, H 10.110, P
3.61%, N 1.95%, Si 0.42% C1 0.80%, found C 57.88%, H
10.20%, P 3.30%, N 1.84%, Si 0.12% C1 0.78%; 'Hnmr (400 MHz, ppm, CD30D:CDC13 1:1 v:v) 4.33, 4.29, 3.97, 3.71, 3.38, 20 3.34, 3.29, 3.22, 1.67, 1.32, 0.92, 0.09; specific viscosity in a lOmg/ml solution of ethanol: chloroform (1:1) is 0.32.
Example 4 Preparation of Polv(2-Methacrylo~loxyethvl)-2' (Trimethylammoniumethvl) Phosphate Inner Salt)-co-n Dodecyl methacrylate)-co-(2-Methacryloyoxy) ethyl trimethyl ammonium chloride)-co-(hydroxY bropyl methacrylate)-co-l3 Trimethoxysilylpropvl methacrvlate) 23'47~6~20'4 polymers 4.1 Monomer Feed Synthesis Zwitterionic monomer (34.1Og, 0.116 mole) and cationic monomer (6.3g, 0.030 mole) were weighed in a glove box environment dried by Pz05. Dodecyl methacrylate (60.O1g, 0.236 mole), hydroxypropyl methacrylate monomer (14.51g, 0.101 mole), trimethoxysilyl monomer (5.OOg, 0.020 mole) and AIBN initiator (0.2409g, 0.2%) were weighed in air. A
3 neck reaction flask, fitted with water condenser, nitrogen gas flow and monomer feed tubing, and primed with anhydrous n-propanol:isopropyl acetate (60:40 mass ratio) solvent, was immersed in a heated 90°C oil bath. The monomers and initiator were dissolved in n-propanol:iso propyl acetate solvent and magnetically stirred in a measuring cylinder_sealed with parafilm. The reaction mixture was drawn into polypropylene tubing placed inside the measuring cylinder and through silicone tubing via a peristaltic pump to enter the heated reaction vessel in a dropwise process. A complete transfer to the heated vessel took 2 hours. The reaction was stirred for another hour.
A second charge of AIBN initiator (0.02418, 0.02wt%) was added and the reaction mixture was stirred for a further hour, taking the total reaction time to 4 hours. Total solids content was 30 wt% in n-propanol:isopropyl acetate (168.068:112.088).
Once cooled to room temperature, the reaction mixture was split into two batches. The first batch of reaction mixture (240m1) was precipitated by dropwise addition to vigorously stirred methyl acetate (2000m1). The product was separated by Buchner flask and 113 Whatman wet strengthened filter paper, and dried in a room temperature vacuum oven for up to 24 hours. The product was rapidly frozen by liquid nitrogen, milled into a fine powder and further dried in a room temperature vacuum for 24 hours.
The product (50.678, 81.8% based on mass recovery) was bottled in a brown glass vial and stored at 4°C.
The polymer requires by weight C 62.4%, H 9.9%, P
3.0%, N 1.9%, Si 0.4% C1 0.8%, found C 57.0%, H 9.4%, N
1.7%, P 2.7%; 'Hnmr (400 MH2, ppm, CDjOD:CDC13 1:1 v:v) 4.41, 4.08, 3.83, 3.46, 3.40, 3.34, 2.07, 1.67, 1.43, 1.18, I.04.
The product was subjected to chloride ion assay to establish the rate of inclusion of cationic monomer:
required 5.23 wt%, found 4.66 and 4.71 wt%.
4.2 One Pot Synthesis Zwitterionic monomer (3.988, 1.35 x 10-Z mole) , dodecyl methacrylate monomer (7.0098, 2.76 x 10-Z mole) , cationic monomer (0.7338, 0.35 x 10-Z mole), hydroxypropyl methacrylate (1.6918, 0.67 x lo-' mole) and trimethoxysilyl monomer (0.5858, 0.24 x 10-2 mole) were rinsed into the reaction vessel with 98m1 solvent mixture of 15:85 v:v%
MeOH:EtOH. Anhydrous cationic monomer was predissolved in 3m1 pure MeOH before being rinsed into the reaction vessel.
Dodecyl methacrylate was pre-columned through activated basic alumina (Brockmann 1 ca.150 mesh, 508) before use.
Dry nitrogen gas was bubbled through for 20 minutes to degas the reaction mixture at room temperature before immersing the reaction vessel in an oil bath heated to 67°C. The vessel was heated for 15 minutes prior to AIBN
initiator (0.148, l.lwt%) being rinsed into the reaction mixture with 2m1 solvent mixture. The reaction was magnetically stirred and maintained under a positive pressure nitrogen blanket sufficient to bubble through a mineral oil bubbler. The reaction time was 39.5 hours.
Once cooled to room temperature, the reaction mixture was filtered through sintered glass. The solvent was removed at <40°C by rotary evaporator to give a white foam residue that was later redissolved in 58m1 dichloromethane and added dropwise into vigorously stirred 600m1 acetone.
A white solid product settled from the acetone leaving a slightly cloudy supernatant. The product was separated by Buchner flask and 113 Whatman wet strengthened filter paper, and dried in a room temperature vacuum oven for up to 20 hours. The product was milled, further dried in a room temperature vacuum for 24 hours and weighed to provide a 93.2 wt% yield, bottled in a glass jar and refrigerated.
The polymer requires by weight C 62.41%, H9.91%, P
2.99%, N 1.700, Si 0.47%, C1 0.890, found C 58.45%, H
9.45%, P 2.55%, N 1.65% Si 0.34%, C1 1.06%. IHnmr (400 MHz, ppm, CD30D:CD1~ 1:1 v:v) 4.33, 4.29, 3.97, 3.71, 3.38, 3.34, 3.29, 3.22, 1.67, 1.32, 0.92, 0.09. Specific viscosity of lOmg/ml solution in ethanol is 0.33. The polymer product was subjected to the chloride ion assay to establish the rate of inclusion of cationic monomer;
required 5.24wto, found 5.16wto and 5.26 wt%.
Example 5 Preparation of Poly~2Methacryloyloxyethyl) 2'fTrimethvlammoniumethyl) Phosphate Inner Salt)-co-In Dodecvl methacrvlate)-co-(2Methacryloyloxvl ethyl trimethyl ammonium chloride) 33.3:60:6 7 tergolvmers Monomer Feed Synthesis To anhydrous n-propanol:isopropyl acetate (30.Og:8.0g) solvent mixture at room temperature, zwitterionic monomer (13.58, 4.58 x 10-z mole) dodecyl methacrylate (20.98, 8.23 x 10-Z mole) cationic monomer (2.5g, 1.20 x 10-2 mole) were added. To the mixture, AIBN (0.7g, 0.20 wto), dissolved 4g isopropyl acetate, was added. The stirred mixture was parafilm sealed in a measuring cylinder and dropwise added via a peristaltic pump to stirred anhydrous n-propanol:
isopropyl acetate (27g:20g) solvent mixture immersed in a heated 90°C oil bath under N~ gas flow. Complete transfer took 2 hours. The pump tubing was washed with isopropyl acetate (4g) and n-propanol 4g) into the 90°C reaction mixture. The reaction was stirred for another hour, where upon AIBN, (O.Olg, 0.02wt%) dissolved 2m1 isopropyl acetate, was added, the pump tubing was washed with isopropyl acetate (2g) and the reaction was stirred for a further hour.
The heating was stopped after 4 hours and the reaction mixture was pumped to ethyl acetate (450g) at room temperature followed by a pump line wash of n-propanol (3g). The product was allowed to settle and the supernatant was decanted. Product was dissolved with isopropanol (47g) solvent, pumped to ethyl acetate (720g) for 45 minutes, the pump line washed with isopropanol (6g) and the product allowed to settle. The supernatant was decanted and the product was washed with acetone (160g) by stirring for l0 minutes. The supernatant was decanted and the product was filtered (Whatman I3 wet strengthened paper) with an acetone wash (80g). The product was dried at room temperature in a vacuum deccicator for up to 16 h, weighed (31.6g, 87% yield based on mass recovery) and stored in a brown glass vial at 4°C.
Characterisation lHnmr (400MHz, ppm, CD30D:CDC1~ 1:1 v:v) 4.41, 4.08, 3.83, 3.46, 3.40, 3.34, 2.07, 1.67, 1.43, 1.18; Specific viscosity of lOmg/ml solution in ethanol is 0.26.
The polymer was subjected to chloride ion assay to establish the inclusion of cationic monomer, required 5.23 wt%, found 5.28 and 5.36 wt%.
Example 6 Samples of some of the polymers of examples 1 and 2 were tested for their performance in terms of fibrinogen adsorption and heparin activity. A coating solution of the polymer lOmg/ml in isopropyl alcohol, was made up and used to coat the surface of samples of polyethylene terephthalate (p.e.t.). The p.e.t. sample to be subjected to a fibrinogen assay was a 1 x 3 cm sheet, whilst that to be subjected to a heparin assay was 40 micron arterial filter material. The dried coating was subsequently contacted with heparin solution 50 U/ml in PBS, rinsed first with PBS and then with deionised water and dried.
The polymer/heparin coated substrate was subjected to the fibrinogen and heparin tests mentioned above. The results for the heparin activity and fibrinogen adsorption for the polymers of example 2 are given in Table 2 below.
Furthermore example 1 polymer/heparin coated materials were subjected to a stability test. For this the polymer(example 1)/heparin coated substrates were immersed in 1% serum albumin in phosphate buffered saline for periods in the range 0.5 to 6 hours at 37°C. The treated samples were removed, rinsed first with PBS and then with deionised Water, and the heparin activity measured. The results indicate that there is no significant loss of activity after 6 hours of BSA/PBS incubation, whereas comparative tests carried out on the commercially available Duraflo and Medtronic M-40 surfaces showed very poor stability. The results using the Carmeda Bioactive surface showed equivalent stability.
Examgl a 7 As a further performance test, substrates coated with 5 example 1 polymer, with and without heparin loading, were contacted with heparinised blood 15 U/ml for 60 minutes.
The treated samples were removed, rinsed first with PBS and then with deionised water and the heparin activity measured. The results show that surfaces coated with the l0 polymer with pendant cationic and phosphoryl choline groups attract and bind heparin from blood which contains heparin.
The surfaces were also studied under s.e.m. and no biological deposits (e.g. of platelets, blood cells and protein) were observed, for the heparin loaded sample or 15 the non-heparin loaded sample.
As comparisons, tests were also carried out on three commercially available heparinised surfaces. DuraFlo uses ionically bound heparin; Medtronic M-40 is believed to use ionically bound heparin; Medtronic CBM-40 (Carmeda 20 Bioactive) uses end point attached heparin.
For these experiments, filter samples were incubated at room temperature with 5 ml of phosphate buffered saline (PBS) with or without 1% serum albumin (BSA) or fresh heparinized human blood. After 60 min, the samples were 25 rinsed thoroughly with saline and deionised water and heparin activity was measured.
The results are shown in Table 3.
Before incubation with PBS, the heparin activity on the DurafloII sample was 240 mU/cm' and 33.5 mU/cm- on the Medtronic M40. The Carmeda BioActive Surface heparin appeared to be more stable with BSA, but the initial heparin activity was the lowest of all filters tested.
Previous results have shown that another 20 micron Medtronic filter with Carmeda bonded heparin had only 2,3 mU/cmz.
Procedure Add 0.25 g polymer to 25 ml methanol. Once the material has fully dissolved add 75 ml of distilled water to the polymer/methanol mixture. Adjust the pH of the mixture to fall between 8-9. Add 1.0 ml of potassium chromate (5% in w/v distilled water) by pipette to the flask, and titrated to the first brown/red end-point with standardised 0.01 m silver nitrate solution. Repeat the titration, using 75 ml distilled water, but no polymer sample to obtain a blank reading. The level of cationic methacrylate in the polymer is directly proportional to the chloride ion concentration.
Example 1 Preparation of poly (2(methacryloyloxyethyl)-2' trimethvlammoniuml ethyl phosphate innersalt-co-n-dodecyl methacrylate-co-11 methacryloylundecyl-1-trimet~l ammonium bromide) (40:71:81.
2-(Methacryloyloxyethyl)-2'-(trimethyl ammonium) ethyl phosphate inner salt (2.328, 0.0079 mole), n-dodecyl methacrylate (3.618, 0.0142 mole) and 11 methacryloylundecyl-1-trimethyl ammonium bromide (0.598, 0.0016 mole synthesised according to reference example 1) were dissolved in 43m1 of propan-2-of and 17m1 of ethyl acetate.
This monomer solution Was thoroughly degassed by bubbling dry nitrogen gas (dried over molecular sieve) through it for 30 minutes. The initiator, AIBN (0.013608, 0.02 weight % of solution) was then washed into the solution using 3ml of degassed ethanol. The solution was further degassed for five minutes. Maintaining the solution under a slight positive pressure of nitrogen (equivalent to a few ml of mineral oil in a bubbler) the solution was heated to 62°C and stirred vigorously for around 46 hours.
After this time the reaction mixture was allowed to cool to around 40°C before removing all of the solvent using a rotary evaporator under vacuum and at about 40°C
giving a solid foam.
This foam was then dissolved in 24m1 of dichloromethane and precipitated dropwise into an excess, 200m1, of acetone. The product was collected on a Buchner filter funnel and washed with 3 further 20m1 quantities of acetone. The white solid was dried in a vacuum oven for 16 hours at 40°C and weighed.
The resulting polymer, obtained in 83% yield, was a white solid.
'HNMR (400MHz, d, ppm, CD,OD/CDC13) 4.31(b), 4.21(b), 4.07(b), 3.98(b), 3.72(b),3.37, 3.33, 3.29(s), 3.22, 3.17, 1.95, 1.84(b), 1.67(b), 1.33(s), 1.06(b), 0.93(s), C., NMR
(500MHz, d, ppm, CD30D/CDCI3) 176.37, 66.91, 65.90, 63.68, 60.05, 54.50, 53.37, 45.54, 32.69, 30.44, 30...13, 28.92, 26.93, 23.41, 17.31, 14.56.
Example 2 Preparation of poly f2(methacrvloyloxyethvl)-2' trimethylammonium) ethyl phosphate innersalt-co-n-dodecvl methacrylate-co-cholinemethacrylate Using a similar technique to that used in Example 1, but using choline methacrylate (2-methacryloyloxy-ethyl trimethyl ammonium chloride) in place of 11-methacryloyl undecyl-1-trimethyl ammonium bromide, various polymerisations were carried out. The zwitterionic monomer, lauryl (dodecyl) methacrylate monomer and choline methacrylate were mixed at the molar ratio shown in Table 1 below and AIBN as initiator was used at the level shown in the table. The total weight percent of solids in the polymerisation solution is also reported in the table, since it was varied between examples.
The polymers were recovered by essentially the same method as in claim 1 although including an extra dissolution and precipitation step to remove lower molecular weight polymer.
The polymer product was subjected to chloride ion determination to establish the rate of inclusion of cationic monomer into the product. Also some rough molecular weight determinations were carried out.
Example 3 Preparation of Poly(2-(Methacrylovloxyethyl)-2' (Trimethvlammoniumethyl) Phosphate Inner Salt)-co-(n Dodecyl methacrylate)-co-(2-(Methacrylo,Yloxy) ethyl trimethyl ammonium chloride)-co-(3-Trimethyoxysilylprop3rl methacrvlate) 30:60:6:4 terpolymers 3.1 Monomer Feed Synthesis The zwitterionic monomer (40.68g, 0.138mo1e) and cationic monomer (5.73g,0.0275mo1e) were weighed in a glove box environment dried by P205. Dodecyl methacrylate (69.45g, 0.273mo1e), trimethoxysilyl monomer (4.53g,0.0182mo1e) and a-azo-isobutyronitrile (AIBN) initiator (1.202g, 1%) were weighed in air. A 3 neck reaction flask, fitted with water condenser, nitrogen gas flow and monomer feed tubing, and primed with anhydrous n-propanol (60g) solvent, was immersed in a heated 90°C oil bath. The monomers and initiator were dissolved in 300g of n-propanol solvent and magnetically stirred in a measuring cylinder sealed with parafilm. The reaction mixture was drawn into polypropylene tubing placed inside the measuring cylinder and through silicone tubing via a peristaltic pump to enter the heated reaction vessel in a dropwise process.
A complete transfer to the heated vessel took 2.25 hours.
The reaction was stirred for another hour. A second charge of AIBN initiator (0.12g), dissolved in 3m1 n-propanol, was added and the reaction mixture was stirred for a further 50 min, taking the total reaction time to 4 hours.
Once cooled to room temperature, the reaction mixture was filtered through a sintered glass filter. The solvent was removed at 40°C-50°C by rotary evaporator to give a white foam residue that was later redissolved in 480m1 dichloromethane and 40m1 methanol solvent mixture and dropwise precipitated into 4000m1 acetone. A white solid product settled from the acetone leaving a slightly cloudy supernatant. The product was separated by Buchner flask and 113 Whatman wet strenghtened filter paper, and dried in a room temperature vacuum oven for up to 24 hours prior to a second workup and precipitation in acetone. The product was weighed (82.9g) to provide a 68.9 wt% yield, bottled in a brown glass vial and refridgerated.
Characterisation of Product The polymer requires by weight C 63.08%, H 10.13%, P
3.55%, N 1.93%, Si 0.43% C1 0.81%, found C 58.1%, H 9.98%, P 3.09%, N 1.90%, Si 0.20%, lHnmr (400MHz, ppm, CD,OD:CDC1, 1:1 v:v) 4.34, 4.30, 3.98, 3.72, 3.38, 3.29, 3.22, 1.67, 1.32, 0.92, 0.10. Specific viscosity of l0mg/ml solution in ethanol: chloroform (1:1 v:v) is 0.13. The polymer product was subjected to the chloride ion assay to establish the rate of inclusion of cationic monomer;
required 4.76wt%, found 4.82wt% and 4.94wt%.
3.2 One Pot Synthesis Zwitterionic monomer (4.878, 1.65 x 10-' mole), dodecyl methacrylate (8.llg, 3.19 x 10-Z mole), cationic monomer (0.67g, 0.32 x 10-' mole) and trimethoxy-silyl monomer (0.53g, 0.21 x 10-Z mole) were rinsed into the reaction vessel with 114 ml solvent mixture of 15:85 v/v% MeOH:EtOH.
Anhydrous cationic monomer was predissolved in 3m1 pure MeOH before being rinsed into the reaction vessel. Dodecyl methacrylate monomer was pre-columned through activated basic alumina (Brockmann 1 ca.150 mesh, 50g) before use.
Dry nitrogen gas was bubbled through for 20 minutes to degas the reaction mixture at room temperature before immersing the reaction vessel in an oil bath heated to 67°C. The vessel was heated for 15 minutes prior to AIBN
initiator (0.14g) being rinsed into the reaction mixture with 2m1 solvent mixture. The reaction was magnetically stirred and maintained up a positive pressure nitrogen blanket sufficient to bubble through a mineral oil bubbler.
The reaction time was 39 hours.
Once cooled to room temperature, the reaction mixture appeared clear with a slight haze. The solvent was removed 5 at room temperature by rotary evaporator to give a white foam residue that was later redissolved in 50m1 dichloromethane and added dropwise into vigorously stirred 500m1 acetone. A white solid product settled from the acetone leaving a slightly cloudy supernatant. The product l0 was separated by Buchner flask and 113 Whatman wet strengthened filter paper, and dried in a room temperature vacuum oven far up to 72 hours. The product was weighed to provide a 91 wt% yield, bottled in a glass jar and ref rigerated .
15 Characterisation The polymer requires by weight C 62.93%, H 10.110, P
3.61%, N 1.95%, Si 0.42% C1 0.80%, found C 57.88%, H
10.20%, P 3.30%, N 1.84%, Si 0.12% C1 0.78%; 'Hnmr (400 MHz, ppm, CD30D:CDC13 1:1 v:v) 4.33, 4.29, 3.97, 3.71, 3.38, 20 3.34, 3.29, 3.22, 1.67, 1.32, 0.92, 0.09; specific viscosity in a lOmg/ml solution of ethanol: chloroform (1:1) is 0.32.
Example 4 Preparation of Polv(2-Methacrylo~loxyethvl)-2' (Trimethylammoniumethvl) Phosphate Inner Salt)-co-n Dodecyl methacrylate)-co-(2-Methacryloyoxy) ethyl trimethyl ammonium chloride)-co-(hydroxY bropyl methacrylate)-co-l3 Trimethoxysilylpropvl methacrvlate) 23'47~6~20'4 polymers 4.1 Monomer Feed Synthesis Zwitterionic monomer (34.1Og, 0.116 mole) and cationic monomer (6.3g, 0.030 mole) were weighed in a glove box environment dried by Pz05. Dodecyl methacrylate (60.O1g, 0.236 mole), hydroxypropyl methacrylate monomer (14.51g, 0.101 mole), trimethoxysilyl monomer (5.OOg, 0.020 mole) and AIBN initiator (0.2409g, 0.2%) were weighed in air. A
3 neck reaction flask, fitted with water condenser, nitrogen gas flow and monomer feed tubing, and primed with anhydrous n-propanol:isopropyl acetate (60:40 mass ratio) solvent, was immersed in a heated 90°C oil bath. The monomers and initiator were dissolved in n-propanol:iso propyl acetate solvent and magnetically stirred in a measuring cylinder_sealed with parafilm. The reaction mixture was drawn into polypropylene tubing placed inside the measuring cylinder and through silicone tubing via a peristaltic pump to enter the heated reaction vessel in a dropwise process. A complete transfer to the heated vessel took 2 hours. The reaction was stirred for another hour.
A second charge of AIBN initiator (0.02418, 0.02wt%) was added and the reaction mixture was stirred for a further hour, taking the total reaction time to 4 hours. Total solids content was 30 wt% in n-propanol:isopropyl acetate (168.068:112.088).
Once cooled to room temperature, the reaction mixture was split into two batches. The first batch of reaction mixture (240m1) was precipitated by dropwise addition to vigorously stirred methyl acetate (2000m1). The product was separated by Buchner flask and 113 Whatman wet strengthened filter paper, and dried in a room temperature vacuum oven for up to 24 hours. The product was rapidly frozen by liquid nitrogen, milled into a fine powder and further dried in a room temperature vacuum for 24 hours.
The product (50.678, 81.8% based on mass recovery) was bottled in a brown glass vial and stored at 4°C.
The polymer requires by weight C 62.4%, H 9.9%, P
3.0%, N 1.9%, Si 0.4% C1 0.8%, found C 57.0%, H 9.4%, N
1.7%, P 2.7%; 'Hnmr (400 MH2, ppm, CDjOD:CDC13 1:1 v:v) 4.41, 4.08, 3.83, 3.46, 3.40, 3.34, 2.07, 1.67, 1.43, 1.18, I.04.
The product was subjected to chloride ion assay to establish the rate of inclusion of cationic monomer:
required 5.23 wt%, found 4.66 and 4.71 wt%.
4.2 One Pot Synthesis Zwitterionic monomer (3.988, 1.35 x 10-Z mole) , dodecyl methacrylate monomer (7.0098, 2.76 x 10-Z mole) , cationic monomer (0.7338, 0.35 x 10-Z mole), hydroxypropyl methacrylate (1.6918, 0.67 x lo-' mole) and trimethoxysilyl monomer (0.5858, 0.24 x 10-2 mole) were rinsed into the reaction vessel with 98m1 solvent mixture of 15:85 v:v%
MeOH:EtOH. Anhydrous cationic monomer was predissolved in 3m1 pure MeOH before being rinsed into the reaction vessel.
Dodecyl methacrylate was pre-columned through activated basic alumina (Brockmann 1 ca.150 mesh, 508) before use.
Dry nitrogen gas was bubbled through for 20 minutes to degas the reaction mixture at room temperature before immersing the reaction vessel in an oil bath heated to 67°C. The vessel was heated for 15 minutes prior to AIBN
initiator (0.148, l.lwt%) being rinsed into the reaction mixture with 2m1 solvent mixture. The reaction was magnetically stirred and maintained under a positive pressure nitrogen blanket sufficient to bubble through a mineral oil bubbler. The reaction time was 39.5 hours.
Once cooled to room temperature, the reaction mixture was filtered through sintered glass. The solvent was removed at <40°C by rotary evaporator to give a white foam residue that was later redissolved in 58m1 dichloromethane and added dropwise into vigorously stirred 600m1 acetone.
A white solid product settled from the acetone leaving a slightly cloudy supernatant. The product was separated by Buchner flask and 113 Whatman wet strengthened filter paper, and dried in a room temperature vacuum oven for up to 20 hours. The product was milled, further dried in a room temperature vacuum for 24 hours and weighed to provide a 93.2 wt% yield, bottled in a glass jar and refrigerated.
The polymer requires by weight C 62.41%, H9.91%, P
2.99%, N 1.700, Si 0.47%, C1 0.890, found C 58.45%, H
9.45%, P 2.55%, N 1.65% Si 0.34%, C1 1.06%. IHnmr (400 MHz, ppm, CD30D:CD1~ 1:1 v:v) 4.33, 4.29, 3.97, 3.71, 3.38, 3.34, 3.29, 3.22, 1.67, 1.32, 0.92, 0.09. Specific viscosity of lOmg/ml solution in ethanol is 0.33. The polymer product was subjected to the chloride ion assay to establish the rate of inclusion of cationic monomer;
required 5.24wto, found 5.16wto and 5.26 wt%.
Example 5 Preparation of Poly~2Methacryloyloxyethyl) 2'fTrimethvlammoniumethyl) Phosphate Inner Salt)-co-In Dodecvl methacrvlate)-co-(2Methacryloyloxvl ethyl trimethyl ammonium chloride) 33.3:60:6 7 tergolvmers Monomer Feed Synthesis To anhydrous n-propanol:isopropyl acetate (30.Og:8.0g) solvent mixture at room temperature, zwitterionic monomer (13.58, 4.58 x 10-z mole) dodecyl methacrylate (20.98, 8.23 x 10-Z mole) cationic monomer (2.5g, 1.20 x 10-2 mole) were added. To the mixture, AIBN (0.7g, 0.20 wto), dissolved 4g isopropyl acetate, was added. The stirred mixture was parafilm sealed in a measuring cylinder and dropwise added via a peristaltic pump to stirred anhydrous n-propanol:
isopropyl acetate (27g:20g) solvent mixture immersed in a heated 90°C oil bath under N~ gas flow. Complete transfer took 2 hours. The pump tubing was washed with isopropyl acetate (4g) and n-propanol 4g) into the 90°C reaction mixture. The reaction was stirred for another hour, where upon AIBN, (O.Olg, 0.02wt%) dissolved 2m1 isopropyl acetate, was added, the pump tubing was washed with isopropyl acetate (2g) and the reaction was stirred for a further hour.
The heating was stopped after 4 hours and the reaction mixture was pumped to ethyl acetate (450g) at room temperature followed by a pump line wash of n-propanol (3g). The product was allowed to settle and the supernatant was decanted. Product was dissolved with isopropanol (47g) solvent, pumped to ethyl acetate (720g) for 45 minutes, the pump line washed with isopropanol (6g) and the product allowed to settle. The supernatant was decanted and the product was washed with acetone (160g) by stirring for l0 minutes. The supernatant was decanted and the product was filtered (Whatman I3 wet strengthened paper) with an acetone wash (80g). The product was dried at room temperature in a vacuum deccicator for up to 16 h, weighed (31.6g, 87% yield based on mass recovery) and stored in a brown glass vial at 4°C.
Characterisation lHnmr (400MHz, ppm, CD30D:CDC1~ 1:1 v:v) 4.41, 4.08, 3.83, 3.46, 3.40, 3.34, 2.07, 1.67, 1.43, 1.18; Specific viscosity of lOmg/ml solution in ethanol is 0.26.
The polymer was subjected to chloride ion assay to establish the inclusion of cationic monomer, required 5.23 wt%, found 5.28 and 5.36 wt%.
Example 6 Samples of some of the polymers of examples 1 and 2 were tested for their performance in terms of fibrinogen adsorption and heparin activity. A coating solution of the polymer lOmg/ml in isopropyl alcohol, was made up and used to coat the surface of samples of polyethylene terephthalate (p.e.t.). The p.e.t. sample to be subjected to a fibrinogen assay was a 1 x 3 cm sheet, whilst that to be subjected to a heparin assay was 40 micron arterial filter material. The dried coating was subsequently contacted with heparin solution 50 U/ml in PBS, rinsed first with PBS and then with deionised water and dried.
The polymer/heparin coated substrate was subjected to the fibrinogen and heparin tests mentioned above. The results for the heparin activity and fibrinogen adsorption for the polymers of example 2 are given in Table 2 below.
Furthermore example 1 polymer/heparin coated materials were subjected to a stability test. For this the polymer(example 1)/heparin coated substrates were immersed in 1% serum albumin in phosphate buffered saline for periods in the range 0.5 to 6 hours at 37°C. The treated samples were removed, rinsed first with PBS and then with deionised Water, and the heparin activity measured. The results indicate that there is no significant loss of activity after 6 hours of BSA/PBS incubation, whereas comparative tests carried out on the commercially available Duraflo and Medtronic M-40 surfaces showed very poor stability. The results using the Carmeda Bioactive surface showed equivalent stability.
Examgl a 7 As a further performance test, substrates coated with 5 example 1 polymer, with and without heparin loading, were contacted with heparinised blood 15 U/ml for 60 minutes.
The treated samples were removed, rinsed first with PBS and then with deionised water and the heparin activity measured. The results show that surfaces coated with the l0 polymer with pendant cationic and phosphoryl choline groups attract and bind heparin from blood which contains heparin.
The surfaces were also studied under s.e.m. and no biological deposits (e.g. of platelets, blood cells and protein) were observed, for the heparin loaded sample or 15 the non-heparin loaded sample.
As comparisons, tests were also carried out on three commercially available heparinised surfaces. DuraFlo uses ionically bound heparin; Medtronic M-40 is believed to use ionically bound heparin; Medtronic CBM-40 (Carmeda 20 Bioactive) uses end point attached heparin.
For these experiments, filter samples were incubated at room temperature with 5 ml of phosphate buffered saline (PBS) with or without 1% serum albumin (BSA) or fresh heparinized human blood. After 60 min, the samples were 25 rinsed thoroughly with saline and deionised water and heparin activity was measured.
The results are shown in Table 3.
Before incubation with PBS, the heparin activity on the DurafloII sample was 240 mU/cm' and 33.5 mU/cm- on the Medtronic M40. The Carmeda BioActive Surface heparin appeared to be more stable with BSA, but the initial heparin activity was the lowest of all filters tested.
Previous results have shown that another 20 micron Medtronic filter with Carmeda bonded heparin had only 2,3 mU/cmz.
Table 3 shows that the polymer of the invention attracts and binds heparin from the blood sample which had a heparin concentration of 15 U/ml.
Initial results had shown that the coating not loaded with heparin shows heparin activity following incubation with heparin containing human blood (see Table 3).
Two similar arterial filters were coated with the cationic/zwitterionic heparin binding polymer of example 1.
Only one filter was loaded with heparin as described above, the other filter was only washed with PBS. Both filters were perfused in parallel with bovine blood (3.5 L/min) for 6 hrs. The blood contained 644 U heparin/kg. The activated clotting time (measured by the Hemochron method) of the system was 447 sec after 9 min perfusion and fell to 257 sec after 60 min perfusion. After 306 min perfusion, the activated clotting time was 212 sec. Both filters performed similar and showed significantly less blood clots than uncoated filters in similar previous perfusion experiments.
Example 8 The polymer of example 4 was used to coat arterial filter devices. The filter was air plasma treated for 30s prior to coating. In a separate step two dispersions were made up. The first contained 2500U heparin (bovine lung) in PBS (2.5m1) and water (47.5m1). The second contained 250mg polymer in 5om1 isopropylalcohol. The two liquid compositions were mixed together then poured into the plasma treated filter which was shaken vigorously for 15 minutes to ensure contact of all the surfaces of the device with the coating mixture. The mixture was then drained out and the coated device washed three times with water. The rinsed filter was dried and placed in an oven overnight at 50°C to ensure the reactive groups of the polymer had crosslinked.
Example 9 Further samples of polymers of examples 1 and 3 to 5 were coated onto arterial filters using the coating solutions described in example 6. The filters were dip coated with the polymer solutions, which were then dried overnight. The polymers of examples 3 and 4 were kept at 70°C overnight to ensure complete crosslinking. The filters were then tested for their fibrinogen adsorption using the performance test described above. Some samples of filter were, after coating with polymer, were loaded with heparin using the general test described above and then subjected to fibrinogen adsorption and heparin activity tests. The control was untreated filter. Table 4 shows the results for reduction in fibrinogen adsorption as compared to the control and heparin activity for the heparin loaded devices. Comparisons are quoted for two commercially available heparin coatings Medtronic CB-M40, believed to have covalently (end point attached) heparin and Medtronic M-40 believed to have ionically bound heparin, in terms of fibrinogen adsorption and heparin activity. The results show that heparin is adsorbed onto the polymer, the mechanism assumed to be an ion exchange process. The filters coated with the PC polymer have reduced fouling by fibrinogen.
Initial results had shown that the coating not loaded with heparin shows heparin activity following incubation with heparin containing human blood (see Table 3).
Two similar arterial filters were coated with the cationic/zwitterionic heparin binding polymer of example 1.
Only one filter was loaded with heparin as described above, the other filter was only washed with PBS. Both filters were perfused in parallel with bovine blood (3.5 L/min) for 6 hrs. The blood contained 644 U heparin/kg. The activated clotting time (measured by the Hemochron method) of the system was 447 sec after 9 min perfusion and fell to 257 sec after 60 min perfusion. After 306 min perfusion, the activated clotting time was 212 sec. Both filters performed similar and showed significantly less blood clots than uncoated filters in similar previous perfusion experiments.
Example 8 The polymer of example 4 was used to coat arterial filter devices. The filter was air plasma treated for 30s prior to coating. In a separate step two dispersions were made up. The first contained 2500U heparin (bovine lung) in PBS (2.5m1) and water (47.5m1). The second contained 250mg polymer in 5om1 isopropylalcohol. The two liquid compositions were mixed together then poured into the plasma treated filter which was shaken vigorously for 15 minutes to ensure contact of all the surfaces of the device with the coating mixture. The mixture was then drained out and the coated device washed three times with water. The rinsed filter was dried and placed in an oven overnight at 50°C to ensure the reactive groups of the polymer had crosslinked.
Example 9 Further samples of polymers of examples 1 and 3 to 5 were coated onto arterial filters using the coating solutions described in example 6. The filters were dip coated with the polymer solutions, which were then dried overnight. The polymers of examples 3 and 4 were kept at 70°C overnight to ensure complete crosslinking. The filters were then tested for their fibrinogen adsorption using the performance test described above. Some samples of filter were, after coating with polymer, were loaded with heparin using the general test described above and then subjected to fibrinogen adsorption and heparin activity tests. The control was untreated filter. Table 4 shows the results for reduction in fibrinogen adsorption as compared to the control and heparin activity for the heparin loaded devices. Comparisons are quoted for two commercially available heparin coatings Medtronic CB-M40, believed to have covalently (end point attached) heparin and Medtronic M-40 believed to have ionically bound heparin, in terms of fibrinogen adsorption and heparin activity. The results show that heparin is adsorbed onto the polymer, the mechanism assumed to be an ion exchange process. The filters coated with the PC polymer have reduced fouling by fibrinogen.
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i Example Heparin Activity Fibrinogen mU/cm2 reduction 2.5 15.9 79 2.6 11.8 55 2.7 24.3 66 2.8 21.2 74 2.9 36.2 60 2.10 22.1 64 2.11 18.7 67 2.12 35.6 70 2.13 0.9 81 2.14 1.8 57 2.15 0 77 Table 2 Heparin activity in mU/cm2 following incubation with Sample PBS PBS/BSA BLOOD
heparinised DurafloII 25.6 4.8 0 Medtronic 7.1 0.9 0 Medtronic 5 4 Ex. l/ 33.1 18.1 16.4 Heparin Ex 1 - 0 18.3 Table 3 Polymer of Without With Heparin Loading Example Heparia Loading % reduction % reduction Heparin fibrinogen fibrinogen activity MU / cm2 Control 0 100 comparison N/A 56 9 covalently 10 bound Heparin comparison N/A 7 <1 sonically bound 15 Heparin Table 4 Reference Example 1 20 Synthesis of 11-methacryloyl undecyl-1-trimeth~lammonium bromide.
Step 1 To a solution of 11-bromo-1-undecanol (5.05 g, 0.02 mol), triethylamine (2.86 g, 0.028 mol) in dry ethyl 25 acetate (30 ml), a solution of methacryloyl chloride (3.03 g, 0.029 mol) in ethyl acetate (20 ml) was slowly added, and the resulting mixture stirred for 90 min at RT.
The solid was filtered off, and the solvents removed in vacuo to afford predominantly 1-bromo, 11 30 undecylmethacrylate (Yield 6:26 g, 97%). As no starting materials were observed by 1H NMR and TLC Rf 0.69 (chloroform/pet. ether 7:3, v/v), this material was carried through to the second step.
Step 2 The product of step 1 (6.26 g, 0.019 mol) was dissolved in dry acetonitrile (40 ml) and added to a mixture of trimethylamine (2.8 g, 0.047 mol) in acetonitrile (20 ml). The system was purged with nitrogen, and then sealed with a dry ice condenser. The reaction was heated to 50 degrees for 20 hr, and protected from light with aluminium foil.
The remaining trimethylamine was removed on a water pump, and then the solvents removed in vacuo to give an oft-white powder. This was washed with ether (250 ml) and the white solid collected (5.67 g, 76% yield). The ether was evaporated to dryness, and the residue again treated with ether (100 ml) to yield further white solid (1.02 g, 13%). 1H NMR indicated that the desired product was formed.
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i Example Heparin Activity Fibrinogen mU/cm2 reduction 2.5 15.9 79 2.6 11.8 55 2.7 24.3 66 2.8 21.2 74 2.9 36.2 60 2.10 22.1 64 2.11 18.7 67 2.12 35.6 70 2.13 0.9 81 2.14 1.8 57 2.15 0 77 Table 2 Heparin activity in mU/cm2 following incubation with Sample PBS PBS/BSA BLOOD
heparinised DurafloII 25.6 4.8 0 Medtronic 7.1 0.9 0 Medtronic 5 4 Ex. l/ 33.1 18.1 16.4 Heparin Ex 1 - 0 18.3 Table 3 Polymer of Without With Heparin Loading Example Heparia Loading % reduction % reduction Heparin fibrinogen fibrinogen activity MU / cm2 Control 0 100 comparison N/A 56 9 covalently 10 bound Heparin comparison N/A 7 <1 sonically bound 15 Heparin Table 4 Reference Example 1 20 Synthesis of 11-methacryloyl undecyl-1-trimeth~lammonium bromide.
Step 1 To a solution of 11-bromo-1-undecanol (5.05 g, 0.02 mol), triethylamine (2.86 g, 0.028 mol) in dry ethyl 25 acetate (30 ml), a solution of methacryloyl chloride (3.03 g, 0.029 mol) in ethyl acetate (20 ml) was slowly added, and the resulting mixture stirred for 90 min at RT.
The solid was filtered off, and the solvents removed in vacuo to afford predominantly 1-bromo, 11 30 undecylmethacrylate (Yield 6:26 g, 97%). As no starting materials were observed by 1H NMR and TLC Rf 0.69 (chloroform/pet. ether 7:3, v/v), this material was carried through to the second step.
Step 2 The product of step 1 (6.26 g, 0.019 mol) was dissolved in dry acetonitrile (40 ml) and added to a mixture of trimethylamine (2.8 g, 0.047 mol) in acetonitrile (20 ml). The system was purged with nitrogen, and then sealed with a dry ice condenser. The reaction was heated to 50 degrees for 20 hr, and protected from light with aluminium foil.
The remaining trimethylamine was removed on a water pump, and then the solvents removed in vacuo to give an oft-white powder. This was washed with ether (250 ml) and the white solid collected (5.67 g, 76% yield). The ether was evaporated to dryness, and the residue again treated with ether (100 ml) to yield further white solid (1.02 g, 13%). 1H NMR indicated that the desired product was formed.
Claims (22)
1. A terpolymer having an overall cationic charge formed from ethylenically unsaturated monomers comprising a) a zwitterionic monomer of the formula I
YBX ~I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene, or alkylene oligooxa alkylene group any of which optionally has one or more fluorine substituents X is an organic group having a zwitterionic moiety;
and Y is ethylenically unsaturated polymerisable group;
b) a cationic monomer of the formula II
Y1B1Q1 ~~~II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y1 is an ethylenically unsaturated polymerisable group;
and Q1 is an organic group having a cationic moiety; and c) a termonomer of the formula III
Y2H2Q2 ~III
wherein B2 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which may optionally have one or more fluorine substituents;
Y2 is an ethylenically unsaturated polymerisable group;
and Q2 is an organic group having a hydrophobic group selected from alkyl groups having at least six carbon atoms, fluorine substituted alkyl groups and alkyl groups having at least one siloxane substituent.
YBX ~I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene, or alkylene oligooxa alkylene group any of which optionally has one or more fluorine substituents X is an organic group having a zwitterionic moiety;
and Y is ethylenically unsaturated polymerisable group;
b) a cationic monomer of the formula II
Y1B1Q1 ~~~II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y1 is an ethylenically unsaturated polymerisable group;
and Q1 is an organic group having a cationic moiety; and c) a termonomer of the formula III
Y2H2Q2 ~III
wherein B2 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which may optionally have one or more fluorine substituents;
Y2 is an ethylenically unsaturated polymerisable group;
and Q2 is an organic group having a hydrophobic group selected from alkyl groups having at least six carbon atoms, fluorine substituted alkyl groups and alkyl groups having at least one siloxane substituent.
2. A terpolymer according to claim 1 in which Y, Y1 and Y2 are each independently selected from wherein:
R is hydrogen or a C1-C4 alkyl group;
A is -O- or -NR1- where R1 is hydrogen or a C1-C4 alkyl group or R1 is -B-X, B1Q1or B2Q2 where B, B1, B2, Q1, Q2 and X
are as defined in claim 1; and K is a group -(CH2)p OC(O)-, -(CH2)p C(O)O-, -(CH2)p OC(O)O-, -(CH2)p NR2-, -(CH2)p NR2C(O)-, -(CH2)p C(O)NR2-, -(CH2)p NR2C(O)O-, -(CH2)p OC(O)NR2-, -(CH2)p NR2C(O)NR2-, (in which the groups R2 are the same or different) -(CH2)p O-, -(CH2)p SO3 -, or, optionally in combination with B, a valence bond and p is from 1 to 12 and R2 is hydrogen or a C1-C4 alkyl group.
R is hydrogen or a C1-C4 alkyl group;
A is -O- or -NR1- where R1 is hydrogen or a C1-C4 alkyl group or R1 is -B-X, B1Q1or B2Q2 where B, B1, B2, Q1, Q2 and X
are as defined in claim 1; and K is a group -(CH2)p OC(O)-, -(CH2)p C(O)O-, -(CH2)p OC(O)O-, -(CH2)p NR2-, -(CH2)p NR2C(O)-, -(CH2)p C(O)NR2-, -(CH2)p NR2C(O)O-, -(CH2)p OC(O)NR2-, -(CH2)p NR2C(O)NR2-, (in which the groups R2 are the same or different) -(CH2)p O-, -(CH2)p SO3 -, or, optionally in combination with B, a valence bond and p is from 1 to 12 and R2 is hydrogen or a C1-C4 alkyl group.
3. A terpolymer according to claim 1 or claim 2 in which B, B1 and B2 each represent a straight or branched C1-24 alkylene group.
4. A terpolymer according to any one of claims 1 to 3 in which X is an ammonium phosphate ester group.
5. A terpolymer according to any one of claims 1 to 4 in which the zwitterionic monomer has the formula wherein R, A and B are as defined in claim 2;
the groups R3 are the same or different and each is hydrogen C1-24 alkyl, aryl, alkaryl, aralkyl, or two or three of the groups R3 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring; and e is 1 to 6.
the groups R3 are the same or different and each is hydrogen C1-24 alkyl, aryl, alkaryl, aralkyl, or two or three of the groups R3 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring; and e is 1 to 6.
6. A terpolymer according to any one of claims 1 to 5 in which B2 and Q2 together represent a C4-24-alkyl group, optionally having carbon-carbon unsaturated bonds.
7. A terpolymer according to any one of claims 1 to 6 in which Q1 is selected from the group consisting of N+R5 3, P+R5 3 and S+R5 2 in which then R5 groups are the same or different and are selected from hydrogen, C1-4 alkyl and aryl.
8. A terpolymer according to claim 7 in which Q1 is N+(CH3)3.
9. A terpolymer according to anyone of claims 1 to 8 in which the monomers additionally comprise a crosslinkable comonomer of the formula IV
Y3 B3 Q3 ~IV
wherein B3 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally comprises one or more fluorine substituents;
Y3 is an ethylenically unsaturated polymerisable group;
and Q3 is an organic group having a reactive group which, on imposition of an external stimulus, reacts with a coreactive group on the surface of a substrate or which is pendant on the polymer.
Y3 B3 Q3 ~IV
wherein B3 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally comprises one or more fluorine substituents;
Y3 is an ethylenically unsaturated polymerisable group;
and Q3 is an organic group having a reactive group which, on imposition of an external stimulus, reacts with a coreactive group on the surface of a substrate or which is pendant on the polymer.
10. A quater polymer having an overall cationic charge formed from ethylenically unsaturated monomers comprising a) a zwitterionic monomer of the formula I
YBX ~I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene, or alkylene oligooxa alkylene group any of which optionally has one or more fluorine substituents X is an organic group having a zwitterionic moiety;
and Y is ethylenically unsaturated polymerisable group;
b) a cationic monomer of the formula II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y1 is an ethylenically unsaturated polymerisable group;
and Q1 is an organic group having a cationic moiety;
c) a crosslinkable comonomer of the formula IV
wherein B3 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y3 is an ethylenically unsaturated polymerisable group;
and Q3 is an organic group comprising a trialkyoxysilyl group; and d) a coreactive comonomer of the formula IV' Y3' B3' Q3' wherein wherein B3' is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y3' is an ethylenically unsaturated polymerisable group; and is a hydroxyl group.
YBX ~I
wherein B is a bond or a straight or branched alkylene, alkylene-oxa-alkylene, or alkylene oligooxa alkylene group any of which optionally has one or more fluorine substituents X is an organic group having a zwitterionic moiety;
and Y is ethylenically unsaturated polymerisable group;
b) a cationic monomer of the formula II
wherein B1 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y1 is an ethylenically unsaturated polymerisable group;
and Q1 is an organic group having a cationic moiety;
c) a crosslinkable comonomer of the formula IV
wherein B3 is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y3 is an ethylenically unsaturated polymerisable group;
and Q3 is an organic group comprising a trialkyoxysilyl group; and d) a coreactive comonomer of the formula IV' Y3' B3' Q3' wherein wherein B3' is a bond or a straight or branched alkylene, alkylene-oxa-alkylene or alkylene-oligooxa-alkylene group, any of which optionally has one or more fluorine substituents;
Y3' is an ethylenically unsaturated polymerisable group; and is a hydroxyl group.
11. A polymerisation process in which monomers of the formula I, II and III as defined in claim 1 are mixed together and radical polymerisation is initiated whereby a polymer according to claim 1 is formed.
12. A polymerisation process in which monomers of the formula I, II, IV and IV' as defined in claim 10 are mixed together and radical polymerisation is initiated whereby a polymer according to claim 10 is formed.
13. A liquid monomer formulation comprising monomers of the general formula I, II and III as defined in claim 1.
14. A liquid monomer formulation comprising monomers of the general formulae I, II, IV and IV' as defined in claim 10.
15. A liquid coating composition containing a polymer according to any one of claim 1 to 10 suspended or dissolved in a solvent.
16. A composition according to claim 15, further comprising an anionic mucopolysaccharide.
17. A coating process in which the surface of a substrate is coated with a composition according to claim 15 and the solvent is removed.
18. A process according to claim 17 in which the polymer is a terpolymer according to claim 9 or a quater polymer according to claim 10 in which, after the polymer has been coated onto the surface, inter and/or intramolecular crosslinking or reaction with the surface is initiated.
19. A process according to claim 17 or claim 18 in which the coated substrate is subsequently contacted with a solution having suspended or dissolved therein an anionically charged mucopolysaccharide.
20. A process according to claim 19 in which the anionic mucopolysaccharide is selected from heparin, hyaluronic acid, chondroitin sulphate, hirudin and an alginate.
21. Use of a terpolymer according to any one of claims 1 to 10 to increase the biocompatibility of a substrate.
22. The use according to claim 21, wherein the substrate is a blood-contacting device.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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GBGB9624130.2A GB9624130D0 (en) | 1996-11-20 | 1996-11-20 | Biocompatible compositions |
GB9624130.2 | 1996-11-20 | ||
PCT/GB1997/003189 WO1998022516A1 (en) | 1996-11-20 | 1997-11-20 | Biocompatible compositions |
Publications (2)
Publication Number | Publication Date |
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CA2271132A1 CA2271132A1 (en) | 1998-05-28 |
CA2271132C true CA2271132C (en) | 2007-04-17 |
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CA002271132A Expired - Fee Related CA2271132C (en) | 1996-11-20 | 1997-11-20 | Biocompatible compositions |
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US (2) | US6251964B1 (en) |
EP (2) | EP0939779B1 (en) |
JP (2) | JP4295359B2 (en) |
AT (2) | ATE215971T1 (en) |
AU (3) | AU728887B2 (en) |
CA (1) | CA2271132C (en) |
DE (2) | DE69711913T2 (en) |
ES (1) | ES2143301T3 (en) |
GB (1) | GB9624130D0 (en) |
WO (3) | WO1998022516A1 (en) |
ZA (3) | ZA9710468B (en) |
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1996
- 1996-11-20 GB GBGB9624130.2A patent/GB9624130D0/en active Pending
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1997
- 1997-11-20 EP EP97913311A patent/EP0939779B1/en not_active Expired - Lifetime
- 1997-11-20 US US09/117,729 patent/US6251964B1/en not_active Expired - Lifetime
- 1997-11-20 ZA ZA9710468A patent/ZA9710468B/en unknown
- 1997-11-20 JP JP52336298A patent/JP4295359B2/en not_active Expired - Lifetime
- 1997-11-20 JP JP52336498A patent/JP4051414B2/en not_active Expired - Fee Related
- 1997-11-20 DE DE69711913T patent/DE69711913T2/en not_active Expired - Lifetime
- 1997-11-20 ZA ZA9710467A patent/ZA9710467B/en unknown
- 1997-11-20 ZA ZA9710466A patent/ZA9710466B/en unknown
- 1997-11-20 AU AU50608/98A patent/AU728887B2/en not_active Ceased
- 1997-11-20 AU AU50611/98A patent/AU5061198A/en not_active Abandoned
- 1997-11-20 WO PCT/GB1997/003189 patent/WO1998022516A1/en active IP Right Grant
- 1997-11-20 AU AU50610/98A patent/AU5061098A/en not_active Abandoned
- 1997-11-20 DE DE69701319T patent/DE69701319T2/en not_active Expired - Lifetime
- 1997-11-20 ES ES97913308T patent/ES2143301T3/en not_active Expired - Lifetime
- 1997-11-20 AT AT97913311T patent/ATE215971T1/en not_active IP Right Cessation
- 1997-11-20 EP EP97913308A patent/EP0866815B1/en not_active Expired - Lifetime
- 1997-11-20 CA CA002271132A patent/CA2271132C/en not_active Expired - Fee Related
- 1997-11-20 US US09/308,496 patent/US6432314B1/en not_active Expired - Lifetime
- 1997-11-20 WO PCT/GB1997/003192 patent/WO1998022517A1/en active IP Right Grant
- 1997-11-20 AT AT97913308T patent/ATE189899T1/en not_active IP Right Cessation
- 1997-11-20 WO PCT/GB1997/003191 patent/WO1998022162A1/en active Application Filing
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CA2271132A1 (en) | 1998-05-28 |
DE69701319D1 (en) | 2000-03-30 |
GB9624130D0 (en) | 1997-01-08 |
JP2001506915A (en) | 2001-05-29 |
EP0866815A2 (en) | 1998-09-30 |
AU5061198A (en) | 1998-06-10 |
JP4295359B2 (en) | 2009-07-15 |
ZA9710466B (en) | 1998-11-20 |
WO1998022162A1 (en) | 1998-05-28 |
AU5060898A (en) | 1998-06-10 |
AU5061098A (en) | 1998-06-10 |
ES2143301T3 (en) | 2000-05-01 |
WO1998022517A1 (en) | 1998-05-28 |
DE69701319T2 (en) | 2000-07-13 |
DE69711913T2 (en) | 2002-10-10 |
ZA9710467B (en) | 1998-11-20 |
EP0866815A3 (en) | 1998-10-21 |
JP2001504877A (en) | 2001-04-10 |
US6432314B1 (en) | 2002-08-13 |
JP4051414B2 (en) | 2008-02-27 |
ATE215971T1 (en) | 2002-04-15 |
EP0866815B1 (en) | 2000-02-23 |
WO1998022516A1 (en) | 1998-05-28 |
ZA9710468B (en) | 1998-11-20 |
EP0939779B1 (en) | 2002-04-10 |
EP0939779A1 (en) | 1999-09-08 |
AU728887B2 (en) | 2001-01-18 |
DE69711913D1 (en) | 2002-05-16 |
US6251964B1 (en) | 2001-06-26 |
ATE189899T1 (en) | 2000-03-15 |
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